Investigation of chemical reactivity of 2-alkoxy-1,4-naphthoquinones and their anticancer activity

Article abstract of DOI:10.1016/j.bmcl.2018.04.060

To establish the structure-activity relationship of 5-hydroxy-1,4-naphthoquinones toward anticancer activity, a series of its derivatives were prepared and tested for the activity (IC₅₀ in μM) against three cell lines; colo205 (colon adenocarcinoma), T47D (breast ductal carcinoma) and K562 (chronic myelogenous leukemia). Among them 2 (IC₅₀: 2.3; 2.0; 1.4 μM), 6 (IC₅₀: 1.9; 2.2; 1.3 μM), 9 (IC₅₀: 0.7; 1.7; 0.9 μM) and 10 (IC₅₀:1.7; 1.0; 1.2 μM) showed moderate to excellent activity. Our perception toward the DNA substitution of alkoxy groups at the C2 position of these naphthoquinones for the anticancer activity led us to investigate their reactivity of substitution toward dimethylamine as a nucleophile. The ease of the substitution of alkoxy groups at the C2 position with dimethylamine is strongly accelerated by hydroxyl group at C5 position and is well correlated with the found anticancer activity results.

Full text of DOI:10.1016/j.bmcl.2018.04.060

Accepted Manuscript
Investigation of Chemical Reactivity of 2-Alkoxy-1,4-naphthoquinones and
their Anticancer Activity
Manoj Manickam, Pulla Reddy Boggu, Jungsuk Cho, Yeo Jin Nam, Seung Jin
Lee, Sang-Hun Jung
PII:
S0960-894X(18)30372-X
https://doi.org/10.1016/j.bmcl.2018.04.060
BMCL 25807
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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22 February 2018
20 April 2018
25 April 2018
Please cite this article as: Manickam, M., Reddy Boggu, P., Cho, J., Nam, Y.J., Lee, S.J., Jung, S-H., Investigation
of Chemical Reactivity of 2-Alkoxy-1,4-naphthoquinones and their Anticancer Activity, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.04.060
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Investigation of Chemical Reactivity of 2-Alkoxy-1,4-naphthoquinones and their
Anticancer Activity
Manoj Manickam, Pulla Reddy Boggu, Jungsuk Cho, Yeo Jin Nam, Seung Jin Lee, Sang-Hun
Jung*
College of Pharmacy and Institute of Drug Research and Development, Chungnam National
University, Daejeon 34134, Korea
Abstract
To establish the structure-activity relationship of 5-hydroxy-1,4-naphthoquinones toward
anticancer activity, a series of its derivatives were prepared and tested for the activity (IC50 in
µM) against three cell lines; colo205 (colon adenocarcinoma), T47D (breast ductal carcinoma)
and K562 (chronic myelogenous leukemia). Among them 2 (IC50: 2.3; 2.0; 1.4 µM), 6 (IC50: 1.9;
2.2; 1.3 µM), 9 (IC50: 0.7; 1.7; 0.9 µM) and 10 (IC50:1.7; 1.0; 1.2 µM) showed moderate to
excellent activity. Our perception toward the DNA substitution of alkoxy groups at the C2
position of these naphthoquinones for the anticancer activity led us to investigate their reactivity
of substitution toward dimethylamine as a nucleophile. The ease of the substitution of alkoxy
groups at the C2 position with dimethylamine is strongly accelerated by hydroxyl group at C5
position and is well correlated with the found anticancer activity results.
Keywords: 2-Alkoxy-5-hydroxy-1,4-naphthoquinone, anticancer activity, chemical reactivity.
*Corresponding author: jungshh@cnu.ac.kr. Tel.; +82 42 821 5939; Fax +82 42 823 6566;
Present address: College of Pharmacy and Institute of Drug Research and development,
Chungnam National University, Daejeon 34134, Korea.

Cancer is a major health problem worldwide and the second most common cause of death.1 The
number of people living beyond a cancer diagnosis reached nearly 14.5 million in 2014 and is
expected to rise to almost 19 million by 2024.1 Screening for new anticancer drugs is still
important and is one of the major goals in medicinal chemistry.2 Mass screening programs of
natural products by the National Cancer Institute have identified the quinone moiety as an
important pharmacophoric element for cytotoxic activity.3 Quinones are widely distributed in
nature,4 and are represented in many clinically used drugs (e.g. doxorubicin, daunorubicin,
mitroxantrone, mitomycin-C (Fig.1)).5,6 Two major mechanisms have been proposed for the
anticancer action of quinones in a variety of cell systems. First, quinones undergo one electron
reduction by enzymes such as microsomal NADPH-cytochrome P-450 reductase or
mitochondrial NADH-ubiquinone oxidoreductase, yielding the corresponding semiquinone
radicals.7,8 Under aerobic conditions, the semiquinone radical then participates in redox cycling
to generate superoxide anion, all of which are believed to be responsible for most of the drug
activity.9,10 Second, quinones are potent electrophiles, capable of reacting with the thiol groups in
proteins as well as GSH.11,12 Depletion of GSH has been associated with menadione-induced
cytotoxicity.11,13 In addition, cytotoxic quinone derivatives were proven to be topoisomerase I
and II inhibitors.14 Among the quinones, 1,4-naphthoquinones have been found to possess a
diverse range of biological activities, in particular, they exhibit potent anticancer activity.15-19 For
example, starting from simple menadione to calothrixins A and B (Fig. 1), they exhibit potent
anticancer activities.20,21 The attachment of variety of functional groups such as amines, esters,
sulfonamides, to the 1,4-naphthoquinone system are proven to improve their anticancer
activities.22-25 In a similar fashion, the fusion of many heterocycles to 1,4-naphthoquinone
system such as furan, pyran etc., have also been synthesized and checked for anticancer

activity.26 An interesting subgroup of 1,4-naphthoquinones is 5-hydroxy-1,4-naphthoquinones.
For e.g. juglone and plumbagin possess anticancer property (Fig. 1).27-30 Some of the esters
attached to 5-hydroxy-1,4-naphthoquinone have also been prepared and showed anticancer
activity.31
Since 5-hydroxy-1,4-naphthoquinones are not well explored for its anticancer activity, we were
interested to design and synthesis a series of 5-hydroxy-1,4-naphthoquinone analogs and
establish its structure-activity relationship.
Figure 1. Anticancer 1,4-naphthoquinones and 5-hydroxy-1,4-naphthoquinones
Scheme 1 represents the preparation of compounds 1-5. Compounds 1 and 4 were prepared
according to the literature procedure with slight modifications.32 The commercially available
juglone (11) was reacted with dimethylamine at -20 °C to obtain 12 as major product and 13 as

minor product. Both the products upon refluxing with 10% HCl yielded the corresponding 1 and
4. Compounds 1 and 4 on methylation with methyl iodide in presence of sodium carbonate
yielded 2 and 5, respectively. The dimethoxy derivative 3 was prepared by the methylation of 2
with methyl iodide using potassium carbonate as base.
Scheme 1. Preparation of compounds 1-5. Reagents and condition: a) Dimethylamine (2M in
THF), toluene, -20 °C, 24 h b) 10% HCl, dioxane, reflux, 5 h c) CH3I, Na2CO3, DMF, RT, 10 h
d) CH3I, K2CO3, DMF, RT, 15 h.
The synthesis of compounds 6-10 is denoted in Scheme 2. The prepared compound 1 (R = OH)
and the commercially available compound 14 (R = H) on reaction with methyl bromoacetate
using sodium carbonate as base afforded the corresponding C2-O-alkylated analogs 6 and 7. The
occurrence of O-alkylation at C2-position of 1 to form 6 was supported by the literature.31
Further it was substantiated by the assignment through NOESY spectrum. The one proton singlet
at 3rd position (H-3) of the naphthoquinone appears at δ 5.99 ppm. The OCH2 proton at 2nd
position appears δ 4.75 ppm and has a correlation with H-3 at δ 5.99 ppm as shown in the
NOESY spectrum (see supporting information).

Scheme 2. Preparation of compounds 6-10. Reagents and condition: a) BrCH2COOCH3,
Na2CO3, DMF, 0 °C to RT, 5 h b) 3M H2SO4, 10 h, RT c) (i) SOCl2, DCM, reflux, 2 h (ii)
NH4OH, 0.5 h, RT d) dimethylamine (2M in THF), DCC, HOBT, THF -5 °C to RT, 8 h
The hydrolysis of the methyl ester of 6 to its carboxylic acid 8 was carried out using 3M sulfuric
acid at ambient temperature. The carboxylic acid 8 was converted to its amide 9 by refluxing
with thionyl chloride to get the acid chloride followed by the reaction of ammonium hydroxide
solution. In another reaction, 8 was reacted with N,N-dicyclohexylcarbodiimide (DCC) and 1-
hydroxybenzotriazole (HOBt) followed by the addition of dimethylamine to get the
dimethylamine analog 10.
Table 1. 5-Hydroxy-1,4-naphthoquinones (1-10) and their anticancer activity
colo205 (colon
adenocarcinoma)
IC50 (µM)
T47D (ductal
carcinoma)
IC50 (µM)
K562 (chronic
myelogenous
leukemia)
No.
Compound
IC50 (µM)
132
>10
2.3
>10
2.0
>10
1.4
2

>10
>10
>10
3
432
>10
>10
1.9
>10
>10
2.2
>10
>10
1.3
5
6
>10
>10
0.7
>10
>10
1.7
>10
>10
0.9
7
8
9
1.7
1.0
1.2
10
The naphthoquinones 1-10 were evaluated for their in vitro inhibitory activities against three
human cancer cell lines (colo205 (colon adenocarcinoma) T47D (ductal carcinoma) and K562
(chronic myelogenous leukemia). Colo205, T47D and K562 cell lines were purchased from the
American Type Culture Collection and regularly tested to confirm the absence of mycoplasma
contamination using an e-Myco™ Mycoplasma PCR detection Kit (iNtRON Biotechnology).
Cells were maintained under RPMI media with 10% fetal bovine serum, 1%
penicillin/streptomycin, and 2 mM L-glutamine. Cells were seeded in 96-well plates, incubated

for 24 h, and then exposed to compounds at the dose of 0.3, 1, 3, and 10 M for 72 h. Cell
viability was measured with CellTiter Glo® assay following to the manufacturer’s instructions
(Promega) and calculated relative to vehicle (0.1% DMSO)-treated controls. IC50 was
determined with standard curve analysis from SigmaPlot ver.12.0 as described previously.33
Doxorubicin (Sigma) was used as positive control. Independent three experiments were
performed, each in triplicate. The results are listed in Table 1 as concentrations required for 50%
growth inhibition (IC50, M).
IC50 values are listed in parentheses below along with this sequence of these cell lines written
here (colo205 (colon adenocarcinoma), T47D (breast ductal carcinoma) and K562 (chronic
myelogenous leukemia).
2,5-Dihydroxynaphthoquinone (1) did not show any activity (>10; >10; >10) whereas the change
of 2-hydroxy group to 2-methoxy group as shown in compound 2 (2.3; 2.0; 1.4) fetched good
activity. The reason could be visualized such that the existence of the tautomerism of the 2-
hydroxy group to its keto group in 1 disfavors the nucleophilic substitution of DNA, whereas in
case of 2, DNA can readily substitute the 2-methoxy group, thus showing anticancer activity.
The change of 5-hydroxy group to 5-methoxy group in compound 2 as shown in compound 3
(>10; >10; >10) abolished the activity which shows the importance of 5-hydroxy group for the
anticancer activity in this naphthoquinone series. The change of 2-hydroxy group to the next
position as shown in compound 4 (>10; >10; >10) or its methylation to methoxy group as shown
in compound 5 (>10; >10; >10) did not show any activity. From these results, it is evident that
the methoxy group and the hydroxy group in the naphthoquinone should be placed in 2 and 5
positions respectively.

In the next series, the 2-methoxy group was changed to 2-methoxycarbonylmethyoxy group as
represented in compound 6 (1.9; 2.2; 1.3) for increasing the electrophilicity of naphthoquinone
moiety toward DNA substitution. The activity was similar to that of compound 2 in all the three
cell lines. The removal of 5-hydroxy group from compound 6 as shown in compound 7 (>10;
>10; >10) abolished the anticancer activity which once again proved the importance of 5-
hydroxy group. The change of the ester group of 6 to carboxylic acid as shown in compound 8
(>10; >10; >10) did not give the activity, probably due to its poor cell permeability. Next the
replacement of carboxylic acid 8 to its amide functionality as demonstrated in compound 9 (0.7;
1.7; 0.9) gained potent activity. Methylation of the amide of 9 to its N,N-dimethyl analog as
shown in compound 10 (1.7; 1.0; 1.2) also retained the activity.
Naphthoquinones were also reported as potential bioreductive alkylating agents and inhibits the
synthesis of DNA in neoplastic cells.34 The nucleophilic substitution of DNA to the C2 position
of the naphthoquinone favored by the hydroxy group at C5 position was envisioned as one of the
reason for the anticancer activity in this naphthoquinone series. To support this view, dimethyl
amine was reacted with the prepared compounds 1-10 and the results are tabulated in Table 2.

Table 2. Reactivities of the naphthoquinones with dimethylamine
Yield
%
No.
1
Reactant
Product
* Reaction
conditions
Time period
no reaction
THF or EtOH,
-10 °C to 60
°C
>48 h
---
THF or EtOH,
-10 °C
10 h
>95
---
2
3
no reaction
no reaction
no reaction
THF or EtOH,
-10 °C to 60
°C
>48 h
THF or EtOH,
-10 °C to 60
°C
>48 h
>48 h
---
---
4
5
THF or EtOH,
-10 °C to 60
°C
THF or EtOH,
-10 °C
2-3 h
24 h
>95
>95
6
7
THF or EtOH,
-10 °C
precipitation due THF or EtOH,
---
---
>95
>95
8
9
to salt formation
-10 °C
THF or EtOH,
-10 °C
30-40 min
30-40 min
THF or EtOH,
-10 °C
10
* Procedure: Naphthoquinones 1-10 was dissolved in THF or EtOH. Dimethylamine (2M inTHF, 1.1equivalent) was
added to the reaction mixture at -10 °C. If the reaction did not proceed, the temperature was gradually increased upto
60 °C. The resulting mixture was evaporated under vacuum and analyzed.

Compound 1 did not react with dimethyl amine even at 60 °C, whereas compound 2 reacted at
-10 °C and the reaction was completed after 10 h. This result indicates that the presence of 2-
methoxy group in 2 favors substitution with dimethyl amine whereas the 2-hydroxy group in 1
tautomerizes to keto form and disfavors the substitution reaction. Similarly compounds 3, 4, 5
did not react with dimethyl amine which clearly denote the importance of location of 5-hydroxy
group from the leaving group to facilitate the substitution reaction at 2nd position holding alkoxy
group such as methoxy group (Fig 2).
Figure 2. Substitution reaction of dimethylamine with naphthoquinone
This statement is further supported by the reaction of compound 6 with dimethylamine which
only took 2-3 h for completion. The importance of 5-hydroxy group for the substitution reaction
at 2nd position was once again proved from the reaction of compound 7 with dimethylamine as it
took 24 h for completion. The reaction of dimethylamine with compound 8 holding a carboxylic
acid group resulted in precipitation, undoubtedly due to salt formation. Finally, the reactions of
compounds 9 and 10 with dimethyl amine resulted in products within 30-40 minutes. This result
proves the presence of amide functionality over ester group favors the substitution reaction at C2
position. The reactivity sequence of the naphthoquinones 1-10 with dimethylamine is well
correlated with its anticancer activity along all the three cell lines.

In conclusion, a series of 5-hydroxy-1,4-naphthoquinone analogs have been prepared to establish
the structure-activity relationship for the anticancer activity (IC50 in µM) against three cell lines;
colo205 (colon adenocarcinoma), T47D (breast ductal carcinoma) and K562 (chronic
myelogenous leukemia). Among them 5-hydroxy-2-methoxynaphthalene-1,4-dione (2, 2.3; 2.0;
1.4 µM), methyl 2-(5-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yloxy)acetate (6, 1.9; 2.2; 1.3
µM), 2-(5-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yloxy)acetamide (9, 0.7; 1.7; 0.9 µM)
and 2-(5-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yloxy)-N,N-dimethylacetamide (10, 1.7;
1.0; 1.2 µM) showed moderate to excellent activity. Hydroxyl group at 5th position is crucial for
the activity. Hydrophobic alkoxyl group at C2 position such as methoxy,
methoxycarbonylmethoxyl or amidomethoxyl is important. Our speculation towards the DNA
substitution at the C2 position of this naphthoquinone for the anticancer activity directed us to
react dimethylamine as a nucleophile with the naphthoquinones 1-10. The ease of the
substitution of the alkoxy groups present in the C2 position of naphthoquinones with
dimethylamine is strongly supported by hydroxyl group at C5 position and is well correlated
with the anticancer activity results (Fig.3). The observed results were considered as preliminary
and the active compounds 9 and 10 can be taken as lead to find more potent 5-hydroxy-2-
substituted naphthoquinones as novel anticancer agents.
Figure 3. Structure activity relationship of 5-hydroxy-1,4-naphthoquinone analogs

Acknowledgment
This work was supported by Priority Research Centers Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology
(2009-0093815).
Supplementary data
The detailed experimental part of this article can be found in the Supplementary data.
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Highlights
 SAR of 1,4-naphthoquinones toward anticancer activity was established.
 C2-Alkoxy and C5-hydroxyl is crucial for activity.


The substitution of alkoxyl at the C2 with amine is accelerated by hydroxyl at C5.
The chemical reactivity of these analogs is correlated with their anticancer activity.

Graphical abstract:
Investigation of Chemical Reactivity of 2-Alkoxy-1,4-naphthoquinones and their
Anticancer Activity