Synthesis and biological evaluation of sulfur-containing shikonin oxime derivatives as potential antineoplastic agents

Article abstract of DOI:10.1016/j.ejmech.2017.11.031

As a continuation of our research on developing potent and potentially safe antineoplastic agents, a set of forty five sulfur-containing shikonin oxime derivatives were synthesized and evaluated for their in vitro cytotoxic activity against human colon cancer (HCT-15), gastric carcinoma (MGC-803), liver (Bel7402), breast (MCF-7) cancer cells and human skin fibroblast (HSF) cells. All the synthesized compounds exhibited potent cytotoxic activity selectively towards HCT-15 cells and did not display apparent toxicity to the normal HSF cells, some of which were more or comparatively effective to the parent compound against HCT-15, MGC-803 and Bel7402 cells. The most active agent 9m displayed high potency against human cancer cells with IC₅₀ ranging from 0.27 ± 0.02 to 9.23 ± 0.12 μM. The structure-activity relationships (SARs) studies suggested that the nature of substituent group in the side chain is important for antitumor potency in vitro. Additionally, nitric oxide release studies revealed that the amount of nitric oxide generated from these oxime derivatives was relatively low. Furthermore, cellular mechanism investigations indicated that compound 9m could arrest cell cycle at G1 phase and induce a strong apoptotic response in HCT-15 cells. Moreover, western blot studies revealed that compound 9m induced apoptosis through the down-regulation of Bcl-2 and up-regulation of Bax, caspase 3 and 9. For all these reasons, compound 9m hold promising potential as antineoplastic agent.

Full text of DOI:10.1016/j.ejmech.2017.11.031

Accepted Manuscript
Synthesis and biological evaluation of sulfur-containing shikonin oxime derivatives as
potential antineoplastic agents
Guang Huang, Hui-Ran Zhao, Qing-Qing Meng, Qi-Jing Zhang, Jin-Yun Dong, Bao-
quan Zhu, Shao-Shun Li
PII:
S0223-5234(17)30934-0
10.1016/j.ejmech.2017.11.031
EJMECH 9906
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 September 2017
Revised Date: 30 October 2017
Accepted Date: 11 November 2017
Please cite this article as: G. Huang, H.-R. Zhao, Q.-Q. Meng, Q.-J. Zhang, J.-Y. Dong, B.-q. Zhu,
S.-S. Li, Synthesis and biological evaluation of sulfur-containing shikonin oxime derivatives as
potential antineoplastic agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.11.031.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of sulfur-containing shikonin oxime
derivatives as potential antineoplastic agents
Guang Huang a, *, Hui-Ran Zhao b, Qing-Qing Meng a, Qi-Jing Zhang a, Jin-Yun Dong a,
Bao-quan Zhu c, Shao-Shun Li a, *
a School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
b School of Pharmaceutical Sciences and Chemistry, Dali University, Dali 671000, China
c Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, P.R. China
Corresponding authors.
E-mail addresses: guanghuangmedchem@hotmail.com (G. Huang), ssli@sjtu.edu.cn (S.-S. Li).
Abstract
As a continuation of our research on developing potent and potentially safe antineoplastic agents,
a set of forty five sulfur-containing shikonin oxime derivatives were synthesized and evaluated for
their in vitro cytotoxic activity against human colon cancer (HCT-15), gastric carcinoma
(MGC-803), liver (Bel7402), breast (MCF-7) cancer cells and human skin fibroblast (HSF)
cells. All the synthesized compounds exhibited potent cytotoxic activity selectively towards HCT-15
cells and did not display apparent toxicity to the normal HSF cells, some of which were more or
comparatively effective to the parent compound against HCT-15, MGC-803 and Bel7402 cells. The
most active agent 9m displayed high potency against human cancer cells with IC50 ranging
from 0.27 ± 0.02 to 9.23 ± 0.12 µM. The structure-activity relationships (SARs) studies
suggested that the nature of substituent group in the side chain is important for antitumor
potency in vitro. Additionally, nitric oxide release studies revealed that the amount of nitric
oxide generated from these oxime derivatives was relatively low. Furthermore, cellular
mechanism investigations indicated that compound 9m could arrest cell cycle at G1 phase and
induce a strong apoptotic response in HCT-15 cells. Moreover, western blot studies revealed
that compound 9m induced apoptosis through the down-regulation of Bcl-2 and up-regulation
of Bax, caspase 3 and 9. For all these reasons, compound 9m hold promising potential as
antineoplastic agent.

ACCEPTED MANUSCRIPT
Keywords
sulfur-containing; shikonin oxime derivatives; anticancer activity; apoptosis
1. Introduction
Currently, many clinical anticancer drugs are either natural products or have been
developed from naturally occurring lead compounds, such as etoposide, vincristine and
docetaxel [1]. Shikonin, one of the most important 1,4-naphthoquinones isolated from the
plant roots of Lithospermum erythrohizon, has attracted growing attention as hallmark
molecule due to its striking anticancer activity [2]. Structural modifications of shikonin have
mainly focused on the hydroxyl groups of the naphthazarin ring and side chain, and thus
many investigations on the synthesis and anticancer activity of hydroxyl-modified shikonin
derivatives have been reported in the past few years [3-12]. However, these derivatives, while
showing comparable or stronger cytotoxic activity than the parent compound in vitro, have
failed to develop as preclinical drug candidates owing to the significant toxicity and lacking
cellular selectivity. This implies that only modifications of hydroxyl groups of the
naphthazarin ring and side chain is not a feasible strategy to overcome drawbacks in the
structure of shikonin itself because naphthazarin scaffold is capable of producing reactive
oxygen species (ROS) and bioreductive alkylation, significantly resulting in the death of
cancer cells and normal cells without distinction.
It was found that oxime is one of the traditional nitric oxide (NO) donors [13] and
meanwhile NO has an anti-neoplastic function including anti-angiogenesis, inhibition of
metastasis and induction of apoptosis in cancer cells [14,15]. Many existing results also
indicated that oximation of the carbonyl group in some compounds, when compared to their
corresponding ketones, could increase several-fold antitumor activity [16]. These led us to
suppose that oximation of the carbonyl group in shikonin could not only reduce the levels of
ROS generation and bioreductive alkylation resulting from the naphthazarin nucleus but retain
its antitumor activity. As expected, our recent investigations demonstrated that shikonin oxime
derivatives displayed more significant cytotoxic activity against cancer cells together with no
apparent cytotoxicity towards normal cells than the parent compound shikonin, and the

ACCEPTED MANUSCRIPT
mechanism of their in vitro potent cytotoxicity was not ascribed to ROS and bioreductive
alkylation [17-20].
On the other hand, our earlier research revealed that the introduction of thioether
functional group at the 1’-position in the side chain of shikonin led to the significant
enhancement of cytotoxicity [8]. In our following study [21], we found that most of
sulfur-containing 6-substituted 5,8-O-dimethyl-1,4-naphthoquinones derivatives showed more
potent cytotoxic activity than that of the corresponding oxygen-containing ones, which was in
line with the evidences that sulfur-containing compounds exhibited significantly higher
biological activity than the oxygen-containing counterparts in some cases [22,23].
Additionally, Lin and co-workers [12] reported that shikonin acetyl-β-D-thio-glycoside
derivatives exhibited excellent inhibitory effect against a series of cancer cell lines.
Accordingly, incorporation of sulfur atom into the side chain of shikonin might confer
desirable biological activity.
Enlightened by above-mentioned investigations and as a continuous study on synthesis
and anticancer activity of shikonin derivatives, we decided to introduce the sulfur atom at the
1’-position and further modify the hydroxyl group at the side chain while retaining the
1,4-naphthoquinone dioxime moiety. Herein, a total of 45 new sulfur-containing shikonin
derivatives have been synthesized with their structure determined by 1H NMR, 13C NMR and
ESI/HRMS. The cytotoxic activity, NO releasing ability and cellular mechanism of these
derivatives were also investigated. Furthermore, the SARs were discussed in detail to gain
further insights into the correlation between anticancer activity and substituent group at the
1’-position along with the chiral structure.
2. Results and discussion
2.1. Chemistry
The synthetic route of the target compounds 6a-6k and 9a-9z was shown in Scheme 1.
Aldehyde 1 was prepared using commercially available 1,5-dihydroxynaphthalene as starting
material [21]. The key intermediate 2 was synthesized by zinc-mediated γ-regioselective
prenylation of aldehyde 1 under room temperature, followed by Oxonia-Cope rearrangement

ACCEPTED MANUSCRIPT
o
at 180 C in hexamethylphosphoric triamide (HMPA). An interesting observation was that
higher temperature significantly promoted the conversion of γ addition product into α addition
product 2. Treatment of compoun 2 with 48% hydrobromic acid and thiourea in ethanol and
succesive hydrolysis of the thiouronium salt intermediate under basic condition afforded thiol
3. Subsequent etherification of 3 with hydrocarbon bromide produced thioether 4a-4k in good
yields, and then oxidation of 4a-4k with cerium (IV) ammonium nitrate (CAN) afforded
6-substituted 1,4-naphthoquinones 5a-5k [21]. Afterwards, condensation of 5a-5k with
o
hydroxylamine hydrochloride at 50 C in the presence of pyridine yielded sulfur-containing
shikonin oxime derivatives 6a-6k [20]. Reaction of thiol 3 with carboxylic acid in the
presence of dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP)
afforded intermediates 7a-7z in excellent yield, which were further treated with CAN to yield
1,4-naphthoquinones 8a-8z. Likewise, the target compunds 9a-9z were prepared by treatment
of corresponding intermediates 8a-8z using the identical method described for compounds
6a-6k. Additionally, it should be pointed out that deprotection of compound 9j under acidic
conditions resulted in the formation of amine hydrochloride 9k.
HO
O
OMe
N
OMe
vi
v
O
OMe S
N
OMe S
R
R
HO
5a-5k
6a-6k
OMe OMe
iii
OMe OMe
OMe OMe
OMe OMe S
ii
R
4a-4k
OMe OMe OH
OMe OMe SH
iv
OMe OMe
2
3
i
R
OMe OMe S
O
7a-7z
OMe OMe
HO
N
N
OMe
O
O
OMe
vi
v
CHO
OMe OMe
OMe S
9a-9z
R
R
OMe S
8a-8z
HO
1
O
O
Scheme 1. Reagents and conditions: i) a. prenyl bromide, Zn, THF, r. t., N2, 2 h; b. HMPA, 180 oC, N2, 2 h,
92%; ii) a. 48% HBr, thiourea, EtOH, reflux, N2,1 h; b. 40% NaOH, reflux, N2, 2 h, 70%; iii) 40% NaOH,

ACCEPTED MANUSCRIPT
KI, RBr, EtOH, reflux, 12 h, 74-85%; iv) RCOOH, DCC, DMAP, CH2Cl2, 24 h, 79-93%; v) CAN, EtOAc,
H2O, 8 min, 21-69%; vi) NH2OH.HCl, C2H5OH, Py, 50 oC, N2, overnight, 63-89%.
The synthesis of title compounds (R or S)-6c and (R or S)-9(f, l, m) was carried out as
depicted in Scheme 2 starting from (R or S)-4-methyl-1-(1,4,5,8-tetramethoxynaphthalen-2-
yl)pent-3-en-1-ol ((R or S)-2 ) that was synthesized according to our previously proposed
procedures [21]. Actually, the synthetic approach of chiral sulfur-containing compounds was
similar to the corresponding sulfur-containing racemic ones. However, many efforts including
asymmetric reduction of prochiral thione, coupling with (R or S)-2 and thiolacetic acid to
afford ethanethioate derivatives with configuration inversions through Mitsunobu reaction and
nucleophilic substitution followed by the reduction with LiAlH4 could not afford chiral thiols
3, possibly due to the instability of thione together with steric effects of naphthalene ring and
side chain. Fortunately, (R or S)-2 was subjected to direct thiolation reaction in the present of
Lawesson’s reagent (LR) to generate the key intermediates 3 with retention of configuration
in satisfactory yield.
HO
O
OMe
N
OMe
v
iv
*
*
O
OMe S
N
OMe
S
R
R
HO
(R or S)-5c
OMe OMe
(R or S)-6c
ii
*
OMe OMe
OMe OMe S
OMe OMe
R
(R or S)-4c
i
*
*
OMe OMe
OMe OMe OH
OMe OMe SH
iii
(R or S)-2
(R or S)-3
*
R
OMe OMe S
O
(R or S)-7(f, l, m)
HO
N
N
OMe
O
O
OMe
v
iv
*
*
OMe S
R
O
R
OMe S
HO
O
(R or S)-9(f, l, m)
(R or S)-8(f, l, m)
o
Scheme 2. Reagents and conditions: i) Lawesson’s reagent, 90 C, N2, 0.15 h, 75%; ii) 40% NaOH, KI,
RBr, EtOH, reflux, 12 h, 83%; iii) RCOOH, DCC, DMAP, CH2Cl2, 24 h, 85-94%; iv) CAN, EtOAc, H2O, 8
min, 54-62%; v) NH2OH.HCl, C2H5OH, Py, 50 oC, N2, overnight, 74-82%.

ACCEPTED MANUSCRIPT
2.2. Biological evaluation
2.2.1 The cytotoxicity of sulfur-containing shikonin oxime derivatives
All prepared sulfur-containing shikonin oxime derivatives were evaluated for their in
vitro cytotoxicity against HCT-15, MGC-803, Bel7402, MCF-7 and HSF cell lines by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay
using shikonin and 5-fluorouracil (5-Fu) as positive reference compounds. The cytotoxic
potency of forty five compounds towards human cell lines was gauged by IC50 value that was
calculated by linear regression analysis of the concentration–response curves generated from
each compound.
As shown in Table 1, the majority of sulfur-containing shikonin oxime derivatives
displayed potent cytotoxic activity towards HCT-15, MGC-803 and Bel7402 cell lines with
more superior or comparative activity to the positive controls (shikonin and 5-Fu), while the
first line anticancer drug 5-Fu showed little inhibitory effects on MGC-803 cells (IC50 > 50
µM). Among these oxime derivatives, the IC50 value of the most effective compound 9m
against HCT-15 cells was as low as 0.27 ± 0.02 µM. Meanwhile, the IC50 value of compound
9m against MGC-803, Bel7402 and MCF-7 cells was found to be 0.86 ± 0.11, 0.45 ± 0.04,
and 9.23 ± 0.12 µM, respectively. Additionally, the similar trend of IC50 values of other
compounds was found among tested tumor cell lines in this study including the strongest
cytotoxicity towards HCT-15 cells, moderate cytotoxicity towards MGC-803, and Bel7402
cells, along with the weakest cytotoxicity towards MCF-7 cells. Furthermore, these
sulfur-containing compounds showed no apparent cytotoxicity against normal cells HSF
(IC50 > 50 µM). These results were of great value since the lead compound shikonin displayed
significant cytotoxic activity towards cancer cells with the IC50 values ranging from 0.86 ±
0.12 to 2.40 ± 0.16 µM, but lack of cellular selectivity between cancer cells and normal cells.
For the first series of compounds bearing thioether moiety 6a-6k, some of them showed
less potent or similar cytotoxicity towards cancer cell lines than the parent compound
shikonin. Meanwhile, the cytotoxic activity of thioether oxime derivatives appeared to be
closely correlated with the size of alkyl chain. It was found that the optimal size of alkyl chain
(R group) for antitumor activity of thiother oxime derivatives was C4-C9 since the smaller or
larger one would lead to the decrease in the cytotoxic activity, and also compound 6c (R =

ACCEPTED MANUSCRIPT
isopentyl) and 6g (R = octyl) showed excellent activity to four cell lines. The introduction of
double bond to isopentyl group afforded compound 6d, leading to the decline of cytotoxic
activity. However, replacement of isopentenyl group into farnesyl moiety gave compound 6 k,
which did not cause a significant reduction in cytotoxicity when compared with compound 6c.
Among the second series of compounds containing alkanoyl moiety 9a-9e, 9e exhibited
the weakest inhibitory potency against four cancer cell lines. Compound 9d was also found to
be approximately 2.5-fold less active against HCT-15 cell line with IC50 values of 1.56 ± 0.27
µM than compound 9c (IC50 = 0.59 ± 0.11 µM). These results therefore provided further
evidence supporting our findings that the introduction of longer alkyl group or unsaturated
alkyl group in the side chain of shikonin, to some extent, could cause the anticancer potency
decrease. Besides, compounds with aliphatic thioester moiety in the side chain, except for 9e,
displayed much stronger cytotoxic activity than those with fatty thioether moiety, especially
for HCT-15 cells. For example, sulfur-containing shikonin derivative 9a containing acetyl
group (IC50 = 1.22 ± 0.10 µM) was approximately 5.5 times more active than the
corresponding compound 6a with ethyl group (IC50 = 7.15 ± 0.24 µM) against HCT-15 cells.
Unlike our previous study [8], substituting the alkyl group with aroyl moiety didn’t suffer
from the loss of inhibitory actions against a panel of cancer cell lines, and compounds 9f, 9g,
9l, 9m, 9p, 9s, 9t and 9v bearing aroyl group exhibited much stronger cytotoxicity towards
HCT-15, MGC-803 and Bel7402 cells than shikonin, implying that a proper increase in steric
effects was beneficial to the enhancement of anticancer activity.
It can be observed from Table 1 that replacement of H atom on the benzoyl moiety
(compound 9f) with methyl, 3,4-dimethoxy and 3,4,5-trimethoxy group used as
electron-donating substituents gave rise to compounds 9g, 9l and 9m respectively, which
caused the minor increase in cytotoxic activity. In contrast, compound 9h with
p-trifluoromethylbenzoyl group showed less 5.5 times and 9 times activity towards HCT-15
and Bel7402 cells than compound 9g and meanwhile compound 9i bearing p-nitrobenzoyl
moiety expressed less 10-fold and 17-fold activity against HCT-15 and Bel7402 cells than
compound 9g. Additionally, we found that the cytotoxicity was increased when the nitro on
the benzoyl moiety was replaced by (tertbutoxycarbonyl)amino (compound 9j) or amino
group (compound 9k). Therefore, it could be concluded that the electron-withdrawing effects

ACCEPTED MANUSCRIPT
of substituents on aroyl moiety might have influence on cytotoxic activity. On the other hand,
the position of substituent on benzoyl moiety seemed to affect its cytotoxicity. Although
compound 9n with ortho-methoxy group displayed similar activity to that of compound 9p
possessing para-methoxy moiety, the m-isomer 9o showed less potent cytotoxic activity than
that of o- and p-isomer. This phenomenon could be also found among compounds containing
fluorinated or chloric substituent on the benzoyl moiety (compounds 9q-9v).
The thioester possessing heteroatom significantly affected the cytotoxicity of oxime
derivatives against various cancer lines. For instance, compounds 9w–9z containing
respective 2’-furoyl, 2’-thenoyl, 3’-picolinoyl and 4’-picolinoyl moiety showed less potent
cytotoxicity with the IC50 values of more than 3.0 µM against Bel7402 cells than compound
9f (IC50 = 1.20 ± 0.09 µM). In addition, both the type and position of hetero-atom seemed to
be important in cytotoxic activity. Compared with other heterocyclic compounds, 9x bearing
thenoyl group expressed the strongest cytotoxic activity with the IC50 values of 1.36 ± 0.11
µM against HCT-15 cells. Meanwhile, compound 9y with 3’-picolinoyl group displayed more
than 2-fold activity than that containing 4’-picolinoyl moiety.
Among the prepared racemic compounds ((R)-6c, (S)-6c, (R)-9f, (S)-9f, (R)-9l, (R)-9l,
(R)-9m, (R)-9m), S-isomers showed slightly stronger cytotoxicity than their corresponding
R-isomers. For example, compound (S)-6c caused significant cytotoxicity at lower
concentration (IC50 = 0.92 ± 0.18, 3.12 ± 0.29, 4.85 ± 0.36, and 26.28 ± 1.10 µM), which was
superior to the cytotoxicity induced by (R)-6c (IC50 = 1.94 ± 0.16, 5.87 ± 0.42, 8.95 ± 0.31,
and 39.36 ± 2.26 µM) in HCT-15, MGC-803, Bel7402 and MCF-7 cells, respectively.
Interestingly, we found that S-isomer displayed equal activity to the corresponding raceme.
Thus these results indicated that the configuration of oxime derivatives, to some degree, could
affect the cytotoxic activity.
Table 1
Cytotoxicity of sulfur-containing shikonin oxime derivatives against four human cancer and one normal
cell lines.

ACCEPTED MANUSCRIPT
Compd.
R
IC50 α (µM) on cell lines
HCT-15
MGC-803
Bel7402
MCF-7
HSF
7.15 ± 0.24
4.37 ± 0.13
1.14 ± 0.11
12.51 ± 0.93
9.32 ± 0.46
3.36 ± 0.24
20.13 ± 1.16
12.55 ± 0.95
5.43 ± 0.21
44.25 ± 2.73 > 50
35.98 ± 1.21 > 50
27.17 ± 0.18 > 50
6a
6b
6c
3.80 ± 0.10
7.05 ± 0.18
12.57 ± 0.68
33.73 ± 1.05 > 50
6d
3.46 ± 0.13
1.97 ± 0.11
1.25 ± 0.04
4.09 ± 0.17
7.43 ± 0.25
6.73 ± 0.36
5.51 ± 0.27
2.29 ± 0.12
9.86 ± 0.33
19.15 ± 0.29
11.65 ± 0.21
8.15 ± 0.15
6.37 ± 0.10
13.85 ± 0.59
25.39 ± 0.73
34.32 ± 0.64
14.73 ± 1.01
3.65 ± 0.24
2.73 ± 0.13
1.65 ± 0.27
34.58 ± 1.70 > 50
30.63 ± 2.16 > 50
25.71 ± 0.97 > 50
37.84 ± 2.05 > 50
48.34 ± 1.04 > 50
6e
6f
6g
6h
6i
14.72 ± 0.48 31.59 ± 1.56
> 50
> 50
6j
5.96 ± 0.38
1.22 ± 0.10
1.35 ± 0.11
0.59 ± 0.11
9.98 ± 0.75
2.49 ± 0.23
3.06 ± 0.19
1.49 ± 0.18
37.43 ± 1.46 > 50
23.47 ± 0.59 > 50
26.11 ± 0.96 > 50
16.32 ± 0.62 > 50
6k
9a
9b
9c
1.56 ± 0.27
3.96 ± 0.31
4.53 ± 0.08
29.92 ± 0.19 > 50
9d
9e
9f
7.21 ± 0.76
0.63 ± 0.05
16.87 ± 0.91
1.28 ± 0.12
22.43 ± 0.17
1.20 ± 0.09
45.73 ± 3.05 > 50
17.37 ± 0.34 > 50
9g
9h
9i
0.47 ± 0.04
2.75 ± 0.39
5.09 ± 0.68
0.92 ± 0.05
5.97 ± 0.68
9.30 ± 1.31
0.72 ± 0.03
7.12 ± 0.56
12.58 ± 0.52
15.81 ± 0.12 > 50
30.54 ± 1.63 > 50
44.67 ± 1.17 > 50

ACCEPTED MANUSCRIPT
9j
9k
9l
3.39 ± 0.14
1.68 ± 0.11
0.39 ± 0.05
6.56 ± 0.67
2.45 ± 0.09
1.17 ± 0.13
9.17 ± 0.53
1.26 ± 0.10
0.66 ± 0.03
32.35 ± 2.39 > 50
19.36 ± 0.59 > 50
14.90 ± 0.28 > 50
9m
0.27 ± 0.02
0.86 ± 0.11
0.45 ± 0.04
9.23 ± 0.12
> 50
9n
9o
0.89 ± 0.14
1.67 ± 0.07
1.37 ± 0.06
4.25 ± 0.43
1.06 ± 0.10
5.54 ± 0.19
19.83 ± 0.55 > 50
35.19 ± 2.06 > 50
9p
9q
0.61 ± 0.03
0.62 ± 0.02
0.93 ± 0.15
1.52 ± 0.18
1.09 ± 0.24
1.34 ± 0.15
17.05 ± 0.12 > 50
23.02 ± 0.83 > 50
9r
2.71 ± 0.42
6.47 ± 0.86
8.90 ± 1.02
38.91 ± 1.47 > 50
9s
9t
0.70 ± 0.10
0.53 ± 0.12
1.28 ± 0.08
1.69 ± 0.15
0.89 ± 0.15
0.87 ± 0.21
20.48 ± 1.13 > 50
19.15 ± 1.24 > 50
9u
2.64 ± 0.09
8.73 ± 0.22
12.11 ± 0.19
42.86 ± 0.97 > 50
9v
9w
9x
9y
0.82 ± 0.11
1.98 ± 0.24
1.36 ± 0.11
3.45 ± 0.08
1.45 ± 0.17
5.01 ± 0.39
2.75 ± 0.07
7.56 ± 0.81
1.16 ± 0.05
7.30 ± 0.16
3.04 ± 0.13
10.19 ± 0.46
20.35 ± 0.14 > 50
28.29 ± 1.15 > 50
23.82 ± 0.18 > 50
37.52 ± 0.76 > 50
9z
7.32 ± 0.34
19.45 ± 0.67
26.8 ± 0.53
> 50
> 50

ACCEPTED MANUSCRIPT
(R)-6c
(S)-6c
(R)-9f
(S)-9f
(R)-9l
1.94 ± 0.16
0.92 ± 0.18
1.06 ± 0.10
0.59 ± 0.03
0.84 ± 0.12
5.87 ± 0.42
3.12 ± 0.29
2.67 ± 0.28
1.26 ± 0.13
1.57 ± 0.16
8.95 ± 0.31
4.85 ± 0.36
2.97 ± 0.19
1.48 ± 0.15
1.16 ± 0.11
39.36 ± 2.26 > 50
26.28 ± 1.10 > 50
25.52 ± 0.92 > 50
18.13 ± 0.69 > 50
19.46 ± 0.42 > 50
(S)-9l
0.38 ± 0.09
0.57 ± 0.10
1.12 ± 0.14
1.18 ± 0.11
0.85 ± 0.07
0.97 ± 0.05
15.17 ± 0.78 > 50
12.76 ± 1.23 > 50
(R)-9m
(S)-9m
0.34 ± 0.05
0.97 ± 0.12
0.63 ± 0.10
11.84 ± 1.19 > 50
0.86 ± 0.12
7.53 ± 0.76
1.51 ± 0.10
> 50
1.24 ± 0.08
2.40 ± 0.16
1.25 ± 0.12
Shikonin
5-Fu
20.93 ± 1.07
28.97 ± 1.75 > 50
α IC50 values were calculated from three independent experiments.
2.2.2 Nitric oxide release assay
To evaluate whether newly synthesized oxime derivatives could release nitric oxide, we
determined the percentage of NO released in vitro from bioactive molecules 6c, 9l, 9m, 9p by
using Griess assay according to the stand curve [24]. It should be pointed out that
N-acetylcysteine that contained reduction thiol group played an important role in NO
production from some NO donors. As shown in Table 2, these oxime derivatives released NO
after 4 h, and compound 9m released the highest amount. However, the quantity of NO
generated from sulfur-containing oxime derivatives was much lower than that of previously
reported NO-donating hybrids [24]. This result was possibly attributed to the intermolecular
hydrogen bond effects between the oxime OH group and the oxygen or sulfur atom.
Table 2
In vitro NO released from targeting compounds (%, 2 mol/mol)

ACCEPTED MANUSCRIPT
Compd. % NO release in 0.5 mM N-acetylcysteine
1 h
2 h
3 h
4 h
5 h
6 h
0.023 ± 0.003
0.041 ± 0.005
0.049 ± 0.002
0.038 ± 0.006
0.027± 0.004 0.033 ± 0.002 0.038 ± 0.004 0.029 ± 0.001 0.024 ± 0.002
0.050 ± 0.006 0.063 ± 0.011 0.079 ± 0.008 0.073 ± 0.004 0.065 ± 0.001
0.063 ± 0.005 0.076 ± 0.003 0.091± 0.012 0.084 ± 0.005 0.079 ± 0.007
0.045 ± 0.003 0.058 ± 0.007 0.064 ± 0.002 0.059 ± 0.010 0.053 ± 0.005
6c
9l
9m
9p
2.2.3. Cell cycle arrest effects of 9m on HCT-15 cells
To further investigate the mechanism of cellular cytotoxicity, we examined the effect of
9m on cell cycle distribution by using propidium iodide (PI) staining. As shown in Fig. 1A-B,
the percentage of cells in the G1 phase after the treatment with compound 9m at indicated
concentrations (0.5 or 1.0 µM) were 44.1% and 49.9% when compared with the control
(36.7%), whereas the proportion of cells at S and G2/M phase decreased significantly upon
the application of 9m (p < 0.05 or p < 0.01). These results indicated that sulfur-containing
shikonin derivatives-induced G1 phase of cell cycle arrest might be one of the plausible
mechanisms of cellular cytotoxicity.
Fig.1. Compound 9m induced cell cycle arrest of HCT-15 cells. 9m at indicated concentrations (0, 0.5, and
1.0 µM) was administered to cells seeded in 6-well plates. After 24 h drug treatment, cells were harvested

ACCEPTED MANUSCRIPT
and analyzed by flow cytometry. (A) Diagram of the cell cycle for each phase based on flow cytometry. (B)
The proportions of cells in G1, S, and G2/M phases were calculated. The data were analyzed by Student’s
t-test, *p < 0.05, **p < 0.01.
2.2.4. Apoptosis effects of 9m on HCT-15 cells
It has been previously reported that G1 cell cycle arrest gives rise to apoptosis in various
cancer cells [25]. Accordingly, after investigation on G1 arrest effects of the most active
compound 9m, it was of considerable interest to verify the apoptotic effects induced by the
same compound in HCT-15 cells, including qualitative and quantitative apoptotic cell studies.
HCT-15 cells were incubated with 9m at indicated concentrations (0, 0.5 or 1.0 µM) for 24 h
and then stained with Hoechst 33342, a fluorescent nucleus staining dye. As shown in Fig. 2,
9m treated cells showed typical apoptosis characteristics, such as nuclear fragmentation and
chromatin condensation, and also, 9m treated cells got much stronger fluorescence intensity
than that of untreated control cells. Whereafter, Annexin V/PI staining was used to
quantitatively measure the effect of 9m on HCT-15 cells apoptosis. Cells were treated with
different concentrations of 9m (0, 0.5, 1.0 or 2.0 µM) for 24 h and analyzed for cells changes
in apoptotic markers using flow cytometer. As shown in cytometry apoptosis diagram
(Fig.3A,B), the percentage of apoptotic cells were significantly increased after treatment with
enhancing concentrations of 9m.
Fig.2. Apoptosis of HCT-15 cells detected by Hoechst33342 staining

ACCEPTED MANUSCRIPT
Fig.3. Compound 9m induced apoptosis of HCT-15 cells. (A) HCT-15 cells apoptosis was detected by
Annexin V/PI on flow cytometry. Cells treated with 0, 0.5, 1 and 2 µM 9m for 24 hours were collected and
processed for analysis. (B) The proportion of apoptotic (Annexin V positive) cells increased dependently
with 9m treatment concentration.
2.2.5. The effects on apoptosis-related proteins of 9m-treated HCT-15 cells
Bcl-2 family proteins have been demonstrated to play a crucial role in controlling the
mitochondrial apoptotic pathway, including pro-apoptotic proteins (Bax, Bad) and
anti-apoptotic proteins (Bcl-2, Bcl-xL). Any imbalance of the expression levels of pro- and
anti-apoptotic Bcl-2 family proteins will disrupt the integrity of the outer mitochondrial
membrane, leading to the apoptotic death of cells [26]. To understand the underlying
mechanism of apoptosis induced by 9m, western blot was performed to detect the expression
levels of apoptosis-related proteins. Proteins were extracted from HCT-15 cells treated with
9m at different dose (0, 0.5, 1 and 2 µM). As revealed by the intensities of the
immune-positive bands, the accumulation levels of Bcl-2 were markedly decreased in the
presence of 2 µM 9m, whereas accumulation levels of Bax were increased (Fig. 4A-C),
implying that 9m induces apoptosis by jointly down-regulating Bcl-2 and up-regulating Bax
protein expression levels.
Additionally, the intrinsic and extrinsic pathways were confirmed to activate a series of

ACCEPTED MANUSCRIPT
proteases regarded as caspases that resulted in apoptotic effects. To further probe the
mechanism of 9m-induced apoptosis, we then examined the expression of other
apoptosis-related proteins, such as caspase-3 and -9. As shown in Fig. (4A, D and E), HCT-15
cells treated with 9m showed enhanced levels of the cleaved active forms of caspase-3 and -9
in a dose-dependent manner, suggesting the involvement of caspase activation in the
apoptosis induced by 9m.
Fig.4. Immunodetection of apoptosis related proteins in compound 9m induced apoptosis in HCT-15 cells.
(A) Western blot analysis of Bcl-2, Bax, Caspase-3, and Caspase-9 protein in HCT-15 cells after 24 h
treatment with 9m (0, 0.5, 1.0 and 2.0 µM). (B-E) The ratio of Bcl-2, Bax, Caspase-3, and Caspase-9,
respectively, was analyzed in densitometry by IMAGE J software. Statistical analysis was performed using
Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.005.

ACCEPTED MANUSCRIPT
3. Conclusion
In conclusion, a series of sulfur-containing shikonin oxime derivatives were synthesized
and screened for their cytotoxic activity against four tumor cell lines and one normal
fibroblast cells. All synthesized compounds were found to be safer with lesser cytotoxicity
towards normal cells and meanwhile some oxime derivatives showed more or comparatively
effective to the lead compound, especially for their cytotoxicity selectively towards HCT-15
cells. Among them, compound 9m exhibited the strongest cytotoxicity against three selected
tumor cell lines with IC50 values of 0.27 µM, 0.86 µM and 0.45 µM, respectively. Further
studies to elucidate the cellular cytotoxic mechanism showed that compound 9m could arrest
cell cycle in the G1 phase. Additionally, Hoechst 33342 staining, Annexin V-FITC assay and
Western-blot analysis revealed that compound 9m killed cancer cells by apoptosis through
down-regulating Bcl-2 and up-regulating Bax, caspase 3 and caspase 9 protein expression
levels. However, the amount of nitric oxide release from oxime derivatives was relatively low.
The investigations on the reasons why these oxime derivatives showed excellent selectivity
between cancerous and normal cells are currently underway in our laboratory.
4. Experimental
4.1. Chemistry
Reagents and solvents were obtained from commercial suppliers. Solvents were dried
and purified using standard techniques. Column chromatography was conducted on silica gel
(100−200 mesh) from Qingdao Ocean Chemical Factory. Melting points were determined on
an MPA100 (OptiMelt) Automated Melting Point System. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded on a Varian MERCURY plus-400 spectrometer with TMS
as an internal standard. HRMS spectra were measured on a Waters Q-TOF Premier mass
spectrometer.
4.1.1 Synthesis of the key intermediates was given in the supplementary data.
4.1.2. General Procedures for synthesis of target compounds (6a-6k and 9a-9z)
A mixture of 6-substituted 1,4-naphthoquinone derivatives (0.50 mmol), hydroxylamine
hydrochloride (0.24 g, 3.5 mmol) and pyridine (0.28 g, 3.5 mmol) in anhydrous ethanol (12.0

ACCEPTED MANUSCRIPT
o
mL) was stirred at 50 C for 24 h under nitrogen atmosphere. After cooling, the reaction
mixture was concentrated and then poured into ice-water, and the resulting precipitate was
filtered. The residue was recrystallized from ethanol to give target compounds 6a-6k and
9a-9z.
4.1.2.1 (1E, 4E)-6-(1-(Ethylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (6a)
o
1
Light yellow solid, yield 78%, m.p.: 193–195 C; H NMR (400 MHz, DMSO-d6): δ
12.07 (s, 2H, 2 × (N=OH)), 7.40 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.11 (t, J = 6.8 Hz, 1H,
CH2CH=C), 4.41 (t, J = 7.0 Hz, 1H, ArCHCH2), 3.80 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
2.58 (t, 2H, J = 7.0 Hz, CHCH2CH), 2.38 (q, J = 7.2 Hz, 2H, SCH2CH3), 1.61 (s, 3H,
13
=C-CH3), 1.56 (s, 3H, =C-CH3), 1.13 (t, J = 7.4 Hz, 3H, CH2CH3); C NMR (100 MHz,
DMSO-d6): δ 153.8, 149.1, 147.8, 138.1, 133.4, 124.2, 121.9, 120.1, 119.7, 119.2, 112.5, 61.3,
56.8, 41.6, 34.4, 25.9, 25.1, 18.2, 15.1; HRMS (ESI) m/z calcd. for C20H27N2O4S 391.1692
[M + H]+; found: 391.1690.
4.1.2.2 (1E, 4E)-6-(1-(Butylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (6b)
o
1
Light yellow solid, yield 80%, m.p.: 196–199 C; H NMR (400 MHz, DMSO-d6): δ
12.07 (s, 2H, 2 × (N=OH)), 7.39 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.11 (t, J = 6.8 Hz, 1H,
CH2CH=C), 4.37 (t, J = 7.4 Hz, 1H, ArCHCH2), 3.80 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
2.58 (t, J = 7.0 Hz, 2H, CHCH2CH), 2.43 – 2.34 (m, 2H, SCH2CH2), 1.61 (s, 3H, =C-CH3),
1.56 (s, 3H, =C-CH3), 1.47 – 1.39 (m, 2H, CH2CH2CH2), 1.34 – 1.25 (m, 2H, CH2CH2CH3),
13
0.79 (t, J = 7.2 Hz, 3H, CH2CH3); C NMR (100 MHz, DMSO-d6): δ 153.8, 149.1, 147.8,
138.1, 133.4, 124.3, 121.9, 120.1, 119.7, 119.2, 112.6, 61.3, 56.8, 42.1, 34.5, 31.4, 30.7, 25.9,
21.7, 18.2, 13.9; HRMS (ESI) m/z calcd. for C22H31N2O4S 419.2005 [M + H]+; found:
419.2001.
4.1.2.3 (1E, 4E)-6-(1-(Isopentylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoqui-
none dioxime (6c)
o
1
Light yellow solid, yield 75%, m.p.: 201–203 C; H NMR (400 MHz, DMSO-d6): δ
12.02 (s, 2H, 2 × (N=OH)), 7.35 (s, 2H, QuinH), 7.17 (s, 1H, ArH), 5.07 (t, J = 6.8 Hz, 1H,
CH2CH=C), 4.33 (t, J = 7.2 Hz, 1H, ArCHCH2), 3.75 (s, 3H, OCH3), 3.55 (s, 3H, OCH3),

ACCEPTED MANUSCRIPT
2.54 (t, J = 7.2 Hz, 2H, CHCH2CH), 2.39 – 2.28 (m, 2H, SCH2CH2), 1.56 (s, 3H, =C-CH3),
1.54 – 1.48 (m, 4H, =C-CH3 and CH(CH3)2), 1.31 – 1.25 (m, 2H, SCH2CH2), 0.75 (d, J = 6.6
Hz, 3H, CHCH3), 0.71 (d, J = 6.6 Hz, 3H, CHCH3); 13C NMR (100 MHz, DMSO-d6): δ 153.8,
149.1, 147.8, 138.1, 133.4, 124.3, 121.9, 120.1, 119.7, 112.6, 61.3, 56.8, 42.2, 38.4, 34.5, 29.1,
27.1, 25.9, 22.6, 22.3, 18.2; HRMS (ESI) m/z calcd. for C23H33N2O4S 433.2161 [M + H]+;
found: 433.2159.
4.1.2.4 (1E,4E)-6-(4-Methyl-1-((3-methylbut-2-en-1-yl)thio)pent-3-en-1-yl)-5,8-dimethoxy-1,
4-naphthoquinone dioxime (6d)
o
1
Light yellow solid, yield 81%, m.p.: 205–207 C; H NMR (400 MHz, DMSO-d6): δ
12.02 (s, 2H, 2 × (N=OH)), 7.35 (s, 2H, QuinH), 7.16 (s, 1H, ArH), 5.12 (t, J = 6.6 Hz, 1H,
CH2CH=C), 5.03 (t, J = 7.2 Hz, 1H, SCH2CH=C), 4.31 (t, J = 7.0 Hz, 1H, ArCHCH2), 3.76 (s,
3H, OCH3), 3.55 (s, 3H, OCH3), 3.04 (d, J = 7.2 Hz, 2H, SCH2CH), 2.56 (t, J = 7.0 Hz, 2H,
CHCH2CH), 1.60 (s, 3H, =C-CH3), 1.56 (s, 3H, =C-CH3), 1.51 (s, 3H, =C-CH3), 1.49 (s, 3H,
=C-CH3); 13C NMR (100 MHz, DMSO-d6): δ 153.7, 148.9, 147.8, 137.9, 135.0, 133.3, 124.4,
121.9, 120.8, 120.1, 119.7, 119.2, 112.6, 61.2, 56.8, 41.9, 34.6, 29.2, 26.0, 25.8, 18.2, 17.8;
HRMS (ESI) m/z calcd. for C23H31N2O4S 431.2005 [M + H]+; found: 431.2001.
4.1.2.5 (1E, 4E)-6-(1-(Hexylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (6e)
o
1
Light yellow solid, yield 68%, m.p.: 190–191 C; H NMR (400 MHz, DMSO-d6): δ
12.07 (s, 2H, 2 × (N=OH)), 7.40 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.11 (t, J = 7.2 Hz, 1H,
CH2CH=C), 4.37 (t, J = 7.2 Hz, 1H, ArCHCH2), 3.80 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
2.56 (t, J = 6.8 Hz, 2H, CHCH2CH), 2.44 – 2.38 (m, 2H, SCH2CH2), 1.61 (s, 3H, =C-CH3),
1.56 (s, 3H, =C-CH3), 1.47 – 1.37 (m, 2H, SCH2CH2), 1.26 – 1.11 (m, 6H, 3 × CH2 ), 0.79 (t,
13
J = 7.0 Hz, 3H, CH2CH3); C NMR (100 MHz, DMSO-d6): δ 153.8, 149.1, 147.8, 138.2,
133.3, 124.3, 121.9, 120.1, 119.6, 119.3, 112.6, 61.3, 56.8, 42.2, 34.5, 31.1, 31.1, 29.3, 28.2,
25.9, 22.4, 18.2, 14.3; HRMS (ESI) m/z calcd. for C24H35N2O4S 447.2318 [M + H]+; found:
447.2316.
4.1.2.6 (1E, 4E)-6-(1-(Heptylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoquione
dioxime (6f)
o
1
Light yellow solid, yield 75%, m.p.: 187–189 C; H NMR (400 MHz, DMSO-d6): δ

ACCEPTED MANUSCRIPT
12.03 (s, 2H, 2 × (N=OH)), 7.40 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.11 (t, J = 6.6 Hz, 1H,
CH2CH=C), 4.37 (t, J = 7.2 Hz, 1H, ArCHCH2), 3.80 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
2.58 (t, J = 6.8 Hz, 2H, CHCH2CH), 2.44 – 2.32 (m, 2H, SCH2CH2), 1.61 (s, 3H, =C-CH3),
1.56 (s, 3H, =C-CH3), 1.46 – 1.39 (m, 2H, SCH2CH2), 1.25 – 1.12 (m, 8H, 4 × CH2), 0.81 (t, J
= 6.8 Hz, 3H, CH2CH3); 13C NMR (100 MHz, DMSO-d6): δ 153.8, 149.1, 147.9, 147.8, 138.2,
133.3, 124.3, 121.9, 120.0, 119.6, 119.4, 112.9, 61.3, 56.9, 42.3, 34.6, 31.5, 31.1, 29.3, 28.5,
28.4, 25.9, 22.4, 18.2, 14.3; HRMS (ESI) m/z calcd. for C25H37N2O4S 461.2474 [M + H]+;
found: 461.2469.
4.1.2.7 (1E, 4E)-6-(1-(Octylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (6g)
o
1
Light yellow solid, yield 83%, m.p.: 185–186 C; H NMR (400 MHz, DMSO-d6): δ
12.06 (s, 2H, 2 × (N=OH)), 7.39 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.11 (t, J = 6.6 Hz, 1H,
CH2CH=C), 4.36 (t, J = 7.2 Hz, 1H, ArCHCH2), 3.79 (s, 3H, OCH3), 3.58 (s, 3H, OCH3),
2.57 (t, J = 7.0 Hz, 2H, CHCH2CH), 2.44 – 2.31 (m, 2H, SCH2CH2), 1.60 (s, 3H, =C-CH3),
1.56 (s, 3H, =C-CH3), 1.45 – 1.38 (m, 2H, SCH2CH2), 1.25 – 1.12 (m, 10H, 5 × CH2), 0.82 (t,
13
J = 6.8 Hz, 3H, CH2CH3); C NMR (100 MHz, DMSO-d6): δ 153.8, 149.1, 147.9, 147.8,
138.2, 133.3, 124.3, 121.9, 120.1, 119.6, 119.3, 112.6, 61.3, 56.8, 42.2, 34.6, 31.6, 31.1, 29.3,
29.0, 28.8, 28.5, 25.9, 22.5, 18.2, 14.3; HRMS (ESI) m/z calcd. for C26H39N2O4S 475.2631
[M + H]+; found: 461.2631.
4.1.2.8 (1E, 4E)-6-(1-(Nonylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (6h)
o
1
Light yellow solid, yield 79%, m.p.: 181–183 C; H NMR (400 MHz, DMSO-d6): δ
12.03 (s, 2H, 2 × (N=OH)), 7.40 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.11 (t, J = 6.6 Hz, 1H,
CH2CH=C), 4.37 (t, J = 7.2 Hz, 1H, ArCHCH2), 3.80 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
2.57 (t, J = 7.0 Hz, 2H, CHCH2CH), 2.45 – 2.32 (m, 2H, SCH2CH2), 1.61 (s, 3H, =C-CH3),
1.56 (s, 3H, =C-CH3), 1.46 – 1.38 (m, 2H, SCH2CH2), 1.25 – 1.12 (m, 12H, 6 × CH2), 0.83 (t,
13
J = 6.8 Hz, 3H, CH2CH3); C NMR (100 MHz, DMSO-d6): δ 153.8, 149.1, 147.8, 138.2,
133.3, 124.3, 121.9, 120.0, 119.6, 119.4, 112.9, 61.3, 56.9, 42.4, 34.7, 31.6, 31.1, 29.3, 29.2,
29.0, 28.9, 28.5, 25.9, 22.5, 18.2, 14.3; HRMS (ESI) m/z calcd. for C27H41N2O4S 489.2787
[M + H]+; found: 489.2783.

ACCEPTED MANUSCRIPT
4.1.2.9 (1E, 4E)-6-(1-(Decylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (6i)
o
1
Light yellow solid, yield 86%, m.p.: 174–177 C; H NMR (400 MHz, DMSO-d6): δ
12.02 (s, 2H, 2 × (N=OH)), 7.40 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.11 (t, J = 6.8 Hz, 1H,
CH2CH=C), 4.37 (t, J = 7.2 Hz, 1H, ArCHCH2), 3.80 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
2.60 – 2.53 (m, 2H, CHCH2CH), 2.43 – 2.32 (m, 2H, SCH2CH2), 1.61 (s, 3H, =C-CH3), 1.56
(s, 3H, =C-CH3), 1.45 – 1.38 (m, 2H, SCH2CH2), 1.24 – 1.13 (m, 14H, 7 × CH2), 0.84 (t, J =
6.8 Hz, 3H, CH2CH3); 13C NMR (100 MHz, DMSO-d6): δ 153.8, 149.1, 147.8, 138.2, 133.3,
124.3, 121.9, 120.0, 119.5, 112.8, 61.3, 56.8, 42.4, 34.7, 31.7, 31.1, 29.3, 29.1, 28.9, 28.5,
25.9, 22.5, 18.2, 14.3; HRMS (ESI) m/z calcd. for C28H43N2O4S 503.2944 [M + H]+; found:
503.2941.
4.1.2.10 (1E, 4E)-6-(1-(Dodecylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoqui-
none dioxime (6j)
o
1
Light yellow solid, yield 82%, m.p.: 154–156 C; H NMR (400 MHz, DMSO-d6): δ
12.03 (s, 2H, 2 × (N=OH)), 7.39 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.11 (t, J = 6.8 Hz, 1H,
CH2CH=C), 4.37 (t, J = 7.4 Hz, 1H, ArCHCH2), 3.79 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
2.60 – 2.53 (m, 2H, CHCH2CH), 2.43 – 2.33 (m, 2H, SCH2CH2), 1.61 (s, 3H, =C-CH3), 1.56
(s, 3H, =C-CH3), 1.46 – 1.39 (m, 2H, SCH2CH2), 1.26 – 1.12 (m, 18H, 9 × CH2), 0.85 (t, J =
6.8 Hz, 3H, CH2CH3); 13C NMR (100 MHz, DMSO-d6): δ 153.8, 149.2, 147.8, 138.2, 133.3,
124.3, 121.9, 120.0, 119.5, 112.9, 61.3, 56.8, 42.4, 34.7, 31.7, 31.2, 29.4, 29.3, 29.1, 28.9,
28.5, 25.9, 22.5, 18.2, 14.3; HRMS (ESI) m/z calcd. for C30H47N2O4S 531.3257 [M + H]+;
found: 531.3255.
4.1.2.11 (1E, 4E)-6-(1-(Farnesylthio)-4-methylpent-3-en-1-yl)-5,8-dimethoxy-1,4-naphthoqu-
inone dioxime (6k)
o
1
Light yellow solid, yield 67%, m.p.: 137–140 C; H NMR (400 MHz, DMSO-d6): δ
11.99 (s, 2H, 2 × (N=OH)), 7.35 (s, 2H, QuinH), 7.15 (s, 1H, ArH), 5.11 (t, J = 6.8 Hz, 1H,
CH2CH=C), 5.05 – 4.96 (m, 3H, 3 × (CH2CH=C)), 4.31 (t, J = 7.2 Hz, 1H, ArCHCH2), 3.74
(s, 3H, OCH3), 3.54 (s, 3H, OCH3), 3.08 – 2.99 (m, 2H, CHCH2CH=C)), 2.57 – 2.50 (m, 2H,
SCH2CH2), 2.02 – 1.79 (m, 10H, 5 × CH2), 1.61 – 1.53 (m, 9H, 3 × CH3), 1.51 – 1.46 (m, 9H,
3 × CH3); 13C NMR (100 MHz, DMSO-d6): δ 153.7, 149.0, 147.9, 147.8, 138.5, 138.0, 134.9,

ACCEPTED MANUSCRIPT
133.2, 131.2, 131.0, 125.0, 124.5, 124.4, 124.0, 121.9, 120.5, 120.0, 119.6, 119.4, 112.8, 61.2,
56.8, 42.2, 34.7, 31.9, 29.2, 26.6, 26.4, 26.2, 25.9, 25.8, 23.5, 18.1, 17.9, 17.8, 16.1; HRMS
(ESI) m/z calcd. for C33H47N2O4S 567.3257 [M + H]+; found: 567.3249.
4.1.2.12 (1E, 4E)-6-(1-Acetylthio-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (9a)
o
1
Light yellow solid, yield 70%, m.p.: 184–188 C; H NMR (400 MHz, DMSO-d6): δ
12.09 (s, 2H, 2 × (N=OH)), 7.39 (s, 2H, QuinH), 7.10 (s, 1H, ArH), 5.04 – 4.96 (m, 2H,
CH2CH=C and ArCHCH2), 3.80 (s, 3H, OCH3), 3.61 (s, 3H, OCH3), 2.74 – 2.67 (m, 1H, 1/2
× (CH2CH=C)), 2.64 – 2.57 (m, 1H, 1/2 × (CH2CH=C)), 2.34 (s, 3H, COCH3), 1.59 (s, 6H, 2
13
× (=C-CH3)); C NMR (100 MHz, DMSO-d6): δ 194.8 (C=O), 153.6, 148.7, 147.8, 147.6,
136.0, 134.4, 124.6, 121.1, 120.1, 119.7, 119.6, 113.2, 61.0, 56.9, 41.9, 34.6, 30.8, 25.9, 18.2;
HRMS (ESI) m/z calcd. for C20H25N2O5S 405.1484 [M + H]+; found: 405.1479.
4.1.2.13 (1E, 4E)-6-(1-Acrylthio-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (9b)
o
1
Light yellow solid, yield 63%, m.p.: 185–187 C; H NMR (400 MHz, DMSO-d6): δ
12.07 (s, 2H, 2 × (N=OH)), 7.33 (s, 2H, QuinH), 7.05 (s, 1H, ArH), 5.04 – 4.96 (m, 2H,
CH2CH=C and ArCHCH2), 3.74 (s, 3H, OCH3), 3.55 (s, 3H, OCH3), 2.66 – 2.61 (m, 1H, 1/2
× (CH2CH=C)), 2.57 – 2.51 (m, 3H, 1/2 × (CH2CH=C) and COCH2CH3), 1.54 (s, 6H, 2 ×
13
(=C-CH3)), 1.01 (t, J = 6.8 Hz, 3H, CH2CH3); C NMR (100 MHz, DMSO-d6): δ 199.0,
153.6, 148.7, 147.8, 147.6, 136.1, 134.4, 124.6, 121.1, 120.1, 119.7, 119.6, 113.2, 61.1, 56.9,
41.5, 37.1, 34.6, 25.9, 18.2; HRMS (ESI) m/z calcd. for C21H27N2O5S 419.1641 [M + H]+;
found: 419.1638.
4.1.2.14 (1E, 4E)-6-(1-Isovalerylthio-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphthoquino-
ne dioxime (9c)
o
1
Light yellow solid, yield 76%, m.p.: 192–194 C; H NMR (400 MHz, DMSO-d6): δ
12.05 (s, 2H, 2 × (N=OH)), 7.34 (s, 2H, QuinH), 7.06 (s, 1H, ArH), 5.04 – 4.96 (m, 2H,
CH2CH=C and ArCHCH2), 3.75 (s, 3H, OCH3), 3.56 (s, 3H, OCH3), 2.68 – 2.53 (m, 2H,
CH2CH=C), 2.41 (d, J = 7.2 Hz, 2H, COCH2CH), 2.04 – 1.97 (m, 1H, CH2CH(CH3)2), 1.55 (s,
13
6H, 2 × (=C-CH3)), 0.85 (d, J = 6.4 Hz, 6H, CH(CH3)2); C NMR (100 MHz, DMSO-d6): δ
197.7, 153.6, 148.7, 147.9, 147.7, 136.2, 134.4, 124.7, 121.1, 120.1, 119.8, 119.6, 113.3, 61.0,

ACCEPTED MANUSCRIPT
56.9, 52.4, 41.7, 34.7, 26.6, 25.9, 22.3, 22.3, 18.2; HRMS (ESI) m/z calcd. for C23H31N2O5S
447.1954 [M + H]+; found: 447.1949.
4.1.2.15 (1E, 4E)-6-(1-Senecioylthio-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (9d)
o
1
Light yellow solid, yield 84%, m.p.: 197–199 C; H NMR (400 MHz, DMSO-d6): δ
12.10 (s, 2H, 2 × (N=OH)), 7.39 (s, 2H, QuinH), 7.12 (s, 1H, ArH), 6.05 (s, 1H, COCH=C),
5.10 – 5.00 (m, 2H, CH2CH=C and ArCHCH2), 3.80 (s, 3H, OCH3), 3.62 (s, 3H, OCH3), 2.76
– 2.69 (m, 1H, 1/2 × (CH2CH=C)), 2.66 – 2.57 (m, 1H, 1/2 × (CH2CH=C)), 2.12 (s, 3H,
=C-CH3), 1.87 (s, 3H, =C-CH3), 1.60 (s, 6H, 2 × (=C-CH3)); 13C NMR (100 MHz, DMSO-d6):
δ 187.7, 155.4, 153.6, 148.7, 147.8, 147.7, 136.3, 134.3, 124.7, 122.7, 121.2, 120.1, 119.6,
113.3, 61.0, 56.9, 41.4, 34.8, 27.1, 25.9, 21.4, 18.3; HRMS (ESI) m/z calcd. for C23H29N2O5S
445.1797 [M + H]+; found: 445.1795.
4.1.2.16 (1E, 4E)-6-(1-Hexanoylthio-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (9e)
o
1
Light yellow solid, yield 81%, m.p.: 173–176 C; H NMR (400 MHz, DMSO-d6): δ
12.07 (s, 2H, 2 × (N=OH)), 7.34 (s, 2H, QuinH), 7.06 (s, 1H, ArH), 5.04 – 4.96 (m, 2H,
CH2CH=C and ArCHCH2), 3.75 (s, 3H, OCH3), 3.56 (s, 3H, OCH3), 2.55 – 2.51 (m, 2H,
CH2CH=C), 2.49 – 2.45 (m, 2H, COCH2CH2), 1.58 – 1.52 (m, 8H, 2 × (=C-CH3) and
COCH2CH2), 1.23 – 1.18 (m, 4H, CH2CH2), 0.80 (t, J = 6.8 Hz, 3H, CH2CH3); 13C NMR (100
MHz, DMSO-d6): δ 198.3, 153.6, 148.6, 147.8, 147.6, 136.1, 134.4, 124.6, 121.1, 120.1,
119.6, 113.1, 61.0, 56.9, 43.6, 41.6, 34.7, 30.8, 25.9, 25.3, 22.2, 18.2, 14.2; HRMS (ESI) m/z
calcd. for C24H33N2O5S 461.2110 [M + H]+; found: 461.2113.
4.1.2.17 (1E, 4E)-6-(1-Benzoylthio-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphthoquinone
dioxime (9f)
o
1
Light yellow solid, yield 87%, m.p.: 195–197 C; H NMR (400 MHz, DMSO-d6): δ
12.14 (s, 2H, 2 × (N=OH)), 7.96 (d, J = 7.4 Hz, 2H, ArH), 7.69 (t, J = 6.8 Hz, 1H, ArH), 7.56
(t, J = 7.2 Hz, 2H, ArH), 7.44 (s, 2H, QuinH), 7.25 (s, 1H, ArH), 5.31 (t, J = 7.2 Hz, 1H,
CH2CH=C), 5.11 (t, J = 7.0 Hz, 1H, ArCHCH2), 3.86 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 2.89
13
– 2.71 (m, 2H, CHCH2CH=C), 1.66 (s, 3H, =C-CH3), 1.62 (s, 3H, =C-CH3); C NMR (100
MHz, DMSO-d6): δ 190.7, 153.7, 148.9, 147.9, 147.7, 136.6, 135.8, 134.6, 134.4, 129.5,

ACCEPTED MANUSCRIPT
127.3, 124.8, 121.1, 120.2, 119.9, 119.6, 113.3, 61.1, 57.0, 42.1, 34.6, 25.9, 18.3; HRMS (ESI)
m/z calcd. for C25H27N2O5S 461.1641 [M + H]+; found: 461.1637.
4.1.2.18 (1E, 4E)-6-(1-(4-Methylbenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-napht-
hoquinone dioxime (9g)
o
1
Light yellow solid, yield 89%, m.p.: 198–200 C; H NMR (400 MHz, DMSO-d6): δ
12.12 (s, 2H, 2 × (N=OH)), 7.83 (d, J = 7.6 Hz, 2H, ArH), 7.40 (s, 2H, QuinH), 7.35 (d, J =
7.4 Hz, 2H, ArH), 7.22 (s, 1H, ArH), 5.26 (t, J = 7.0 Hz, 1H, CH2CH=C), 5.08 (t, J = 7.0 Hz,
1H, ArCHCH2), 3.83 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 2.86 – 2.68 (m, 2H, CHCH2CH=C),
13
2.38 (s, 3H, ArCH3), 1.64 (s, 3H, =C-CH3), 1.60 (s, 3H, =C-CH3); C NMR (100 MHz,
DMSO-d6): δ 190.2, 153.6, 148.8, 147.9, 147.7, 145.0, 135.9, 134.6, 134.1, 130.0, 127.4,
124.7, 121.1, 120.1, 119.8, 119.6, 113.3, 61.1, 56.9, 41.9, 34.6, 25.9, 21.6, 18.3; HRMS (ESI)
m/z calcd. for C26H29N2O5S 481.1797 [M + H]+; found: 461.1892.
4.1.2.19 (1E,4E)-6-(1-(4-Trifluoromethylbenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,
4-naphthoquinone dioxime (9h)
o
1
Light yellow solid, yield 85%, m.p.: 173–176 C; H NMR (400 MHz, DMSO-d6): δ
12.14 (s, 2H, 2 × (N=OH)), 8.12 (d, J = 8.2 Hz, 2H, ArH), 7.93 (d, J = 8.0 Hz, 2H, ArH), 7.39
(s, 2H, QuinH), 7.24 (s, 1H, ArH), 5.30 (t, J = 7.4 Hz, 1H, CH2CH=C), 5.09 (t, J = 7.0 Hz, 1H,
ArCHCH2), 3.82 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 2.88 – 2.82 (m, 1H, 1/2 ×
(CHCH2CH=C)), 2.79 – 2.71 (m, 1H, 1/2 × (CHCH2CH=C)), 1.64 (s, 3H, =C-CH3), 1.61 (s,
13
3H, =C-CH3); C NMR (100 MHz, DMSO-d6): δ 190.1, 153.7, 148.9, 147.8, 147.6, 139.8,
135.4, 134.8, 128.2, 126.7, 125.4, 124.7, 122.7, 120.9, 120.1, 119.6, 113.3, 61.2, 57.0, 42.6,
34.4, 25.9, 18.3; HRMS (ESI) m/z calcd. for C26H26F3N2O5S 535.1515 [M + H]+; found:
535.1520.
4.1.2.20 (1E, 4E)-6-(1-(4-Nitrobenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphtho-
quinone dioxime (9i)
o
1
Light yellow solid, yield 83%, m.p.: 188–191 C; H NMR (400 MHz, DMSO-d6): δ
12.11 (s, 2H, 2 × (N=OH)), 8.36 (d, J = 7.6 Hz, 2H, ArH), 8.16 (d, J = 7.4 Hz, 2H, ArH), 7.41
(s, 2H, QuinH), 7.25 (s, 1H, ArH), 5.32 (t, J = 7.4 Hz, 1H, CH2CH=C), 5.11 (t, J = 6.8 Hz, 1H,
ArCHCH2), 3.84 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 2.90 – 2.74 (m, 2H, CHCH2CH=C),
1.65 (s, 3H, =C-CH3), 1.62 (s, 3H, =C-CH3); 13C NMR (100 MHz, DMSO-d6): δ 189.8, 153.7,

ACCEPTED MANUSCRIPT
150.7, 148.9, 147.8, 147.6, 141.2, 135.3, 134.9, 128.7, 124.7, 120.8, 120.1, 119.6, 113.3, 61.2,
57.0, 42.9, 34.4, 25.9, 18.3; HRMS (ESI) m/z calcd. for C25H26N3O7S 512.1491 [M + H]+;
found: 512.1488.
4.1.2.21 (1E,4E)-6-(1-(4-(N-(tert-butoxycarbonyl)amino)benzoylthio)-4-methylpent-3-enyl)-5,
8-dimethoxy-1,4-naphthoquinone dioxime (9j)
o
1
Light yellow solid, yield 85%, m.p.: 237–240 C; H NMR (400 MHz, DMSO-d6): δ
12.02 (s, 1H, 2 × (N=OH)), 9.80 (s, 1H, NH), 7.79 (d, J = 8.0 Hz, 2H, ArH), 7.54 (d, J = 8.0
Hz, 2H, ArH), 7.32 (s, 2H, QuinH), 7.14 (s, 1H, ArH), 5.17 (t, J = 7.4 Hz, 1H, CH2CH=C),
5.00 (t, J = 6.8 Hz, 1H, ArCHCH2), 3.75 (s, 3H, OCH3), 3.57 (s, 3H, OCH3), 2.78 – 2.70 (m,
1H, 1/2 × (CHCH2CH=C)), 2.68 – 2.61 (m, 1H, 1/2 × (CHCH2CH=C)), 1.56 (s, 3H, =C-CH3),
1.53 (s, 3H, =C-CH3), 1.42 (s, 9H, 3 × CH3); 13C NMR (100 MHz, DMSO-d6): δ 189.1, 153.6,
152.8, 148.8, 147.8, 147.6, 145.3, 136.0, 134.5, 130.1, 128.6, 124.7, 121.1, 120.1, 119.8,
119.6, 117.8, 113.3, 80.3, 61.1, 56.9, 41.7, 34.7, 28.4, 25.9, 18.3; HRMS (ESI) m/z calcd. for
C30H36N3O7S 582.2274 [M + H]+; found: 582.2273.
4.1.2.22 (1E, 4E)-6-(1-(4-Aminobenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphth-
oquinone dioxime hydrochloride (9k)
To a stirred solution of compound 9j (72.0 mg, 0.135 mmol) in ethyl acetate (6.5 mL)
was dropwise added hydrochloric acid solution of ethyl acetate (3 M, 0.45 mL). The reaction
mixture was stirred overnight, and the resulting precipitate was filtered. The residue was
recrystallized from ethanol/ethyl acetate to give target compound 9k as pale red solid. Yield
o
1
49%, m.p.: 160–163 C; H NMR (400 MHz, DMSO-d6): δ 7.73 (d, J = 8.6 Hz, 2H, ArH),
7.39 (s, 2H, QuinH), 7.19 (s, 1H, ArH), 6.79 (d, J = 8.6 Hz, 2H, ArH), 6.18 (s, 4H, 2 ×
(N=OH) and NH2), 5.21 (t, J = 7.4 Hz, 1H, CH2CH=C), 5.06 (t, J = 6.8 Hz, 1H, ArCHCH2),
3.81 (s, 3H, OCH3), 3.63 (s, 3H, OCH3), 2.82 – 2.65 (m, 2H, CHCH2CH=C), 1.62 (s, 3H,
13
=C-CH3), 1.59 (s, 3H, =C-CH3); C NMR (100 MHz, DMSO-d6): δ 188.0, 153.6, 148.8,
147.9, 147.6, 136.4, 134.3, 129.6, 126.2, 124.7, 121.3, 120.1, 119.7, 119.6, 115.6, 113.3, 61.1,
56.9, 41.4, 34.9, 28.4, 25.9, 18.3; HRMS (ESI) m/z calcd. for C25H27N3O5S 482.1750 [M –
Cl]+; found: 482.1745.
4.1.2.23 (1E,4E)-6-(1-(3,4-Dimethoxybenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-
naphthoquinone dioxime (9l)

ACCEPTED MANUSCRIPT
o
1
Light yellow solid, yield 83%, m.p.: 197–200 C; H NMR (400 MHz, DMSO-d6): δ
12.09 (s, 2H, 2 × (N=OH)), 7.58 (d, J = 8.6 Hz, 1H, ArH), 7.37 (s, 3H, QuinH and ArH), 7.20
(s, 1H, ArH), 7.05 (d, J = 8.6 Hz, 1H, ArH), 5.22 (t, J = 7.2 Hz, 1H, CH2CH=C), 5.05 (t, J =
7.0 Hz, 1H, ArCHCH2), 3.80 (s, 9H, 3 × OCH3), 3.62 (s, 3H, OCH3), 2.83 – 2.67 (m, 2H,
CHCH2CH=C), 1.61 (s, 3H, =C-CH3), 1.57 (s, 3H, =C-CH3); 13C NMR (100 MHz,
DMSO-d6): δ 189.1, 154.0, 153.7, 149.1, 148.8, 147.9, 147.7, 136.0, 134.6, 129.2, 124.7,
121.9, 121.2, 120.2, 119.8, 119.6, 113.3, 111.6, 109.5, 61.1, 57.0, 56.2, 56.0, 41.8, 34.6, 25.9,
18.3; HRMS (ESI) m/z calcd. for C27H31N2O7S 527.1852 [M + H]+; found: 527.1845.
4.1.2.24 (1E,4E)-6-(1-(3,4,5-Trimethoxybenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4
-naphthoquinone dioxime (9m)
o
1
Light yellow solid, yield 79%, m.p.: 205–206 C; H NMR (400 MHz, DMSO-d6): δ
12.11 (s, 2H, 2 × (N=OH)), 7.40 (s, 2H, QuinH), 7.24 (s, 1H, ArH), 7.20 (s, 2H, ArH), 5.26 (t,
J = 7.2 Hz, 1H, CH2CH=C), 5.08 (t, J = 7.0 Hz, 1H, ArCHCH2), 3.85 (s, 6H, 2 × OCH3), 3.83
(s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.64 (s, 3H, OCH3), 2.88 – 2.81 ((m, 1H, 1/2 ×
(CHCH2CH=C)), 2.79 – 2.70 (m, 1H, 1/2 × (CHCH2CH=C)), 1.66 (s, 3H, =C-CH3), 1.61 (s,
13
3H, =C-CH3); C NMR (100 MHz, DMSO-d6): δ 189.7, 153.7, 153.4, 148.9, 147.7, 142.7,
135.7, 134.7, 131.9, 124.8, 121.1, 120.2, 119.6, 113.3, 104.6, 61.1, 60.6, 57.0, 56.5, 42.0, 34.4,
25.9, 18.3; HRMS (ESI) m/z calcd. for C28H33N2O8S 557.1958 [M + H]+; found: 557.1953.
4.1.2.25 (1E,4E)-6-(1-(2-Methoxybenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-
thoquinone dioxime (9n)
o
1
Light yellow solid, yield 84%, m.p.: 207–209 C; H NMR (400 MHz, DMSO-d6): δ
12.12 (s, 2H, 2 × (N=OH)), 7.68 (d, J = 8.0 Hz, 1H, ArH), 7.48 (d, J = 7.4 Hz, 1H, ArH), 7.41
(s, 2H, QuinH), 7.37 – 7.29 (m, 2H, ArH), 7.21 (s, 1H, ArH), 5.25 (t, J = 7.2 Hz, 1H,
CH2CH=C), 5.09 (t, J = 7.0 Hz, 1H, ArCHCH2), 3.83 (s, 3H, OCH3), 3.67 (s, 3H, OCH3),
2.82 – 2.73 (m, 2H, CHCH2CH=C), 2.40 (s, 3H, OCH3), 1.65 (s, 3H, =C-CH3), 1.63 (s, 3H,
=C-CH3); 13C NMR (100 MHz, DMSO-d6): δ 193.2, 153.6, 148.7, 147.6, 137.5, 136.0, 135.8,
134.5, 132.3, 131.9, 128.1, 126.6, 124.7, 121.1, 120.1, 119.6, 113.2, 61.1, 56.9, 42.4, 34.7,
25.9, 20.2, 18.2; HRMS (ESI) m/z calcd. for C26H29N2O6S 497.1746 [M + H]+; found:
497.1746.
4.1.2.26 (1E,4E)-6-(1-(3-Methoxybenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-

ACCEPTED MANUSCRIPT
thoquinone dioxime (9o)
o
1
Light yellow solid, yield 81%, m.p.: 197–199 C; H NMR (400 MHz, DMSO-d6): δ
12.04 (s, 2H, 2 × (N=OH)), 7.46 (d, J = 7.6 Hz, 1H, ArH), 7.39 (t, J = 8.0 Hz, 1H, ArH), 7.33
(s, 2H, ArH, QuinH), 7.31 (s, 1H, ArH), 7.19 (dd, J = 8.2, 2.6 Hz, 1H, ArH), 7.16 (s, 1H, ArH),
5.19 (t, J = 7.2 Hz, 1H, CH2CH=C), 5.01 (t, J = 6.6 Hz, 1H, ArCHCH2), 3.76 (s, 3H, OCH3),
3.75 (s, 3H, OCH3), 3.58 (s, 3H, OCH3), 2.79 – 2.72 (m, 1H, 1/2 × (CHCH2CH=C)), 2.69 –
13
2.64 (m, 1H, 1/2 × (CHCH2CH=C)), 1.57 (s, 3H, =C-CH3), 1.53 (s, 3H, =C-CH3); C NMR
(100 MHz, DMSO-d6): δ 190.5, 159.9, 153.6, 148.8, 147.6, 138.0, 135.7, 134.6, 130.7, 124.7,
121.0, 120.4, 120.1, 119.8, 119.7, 119.6, 113.2, 111.5, 61.1, 56.9, 55.8, 42.1, 34.5, 25.9, 18.3;
HRMS (ESI) m/z calcd. for C26H29N2O6S 497.1746 [M + H]+; found: 497.1743.
4.1.2.27 (1E,4E)-6-(1-(4-Methoxybenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-
thoquinone dioxime (9p)
o
1
Light yellow solid, yield 83%, m.p.: 201–203 C; H NMR (400 MHz, DMSO-d6): δ
12.03 (s, 2H, 2 × (N=OH)), 7.83 (d, J = 7.2 Hz, 2H, ArH), 7.33 (s, 2H, QuinH), 7.14 (s, 1H,
ArH), 6.98 (d, J = 6.8 Hz, 2H, ArH), 5.18 (t, J = 7.2 Hz, 1H, CH2CH=C), 5.00 (t, J = 6.8 Hz,
1H, ArCHCH2), 3.75 (s, 6H, 2 × OCH3), 3.58 (s, 3H, OCH3), 2.77 – 2.62 (m, 2H,
CHCH2CH=C), 1.55 (s, 3H, =C-CH3), 1.51 (s, 3H, =C-CH3); 13C NMR (100 MHz,
DMSO-d6): δ 189.0, 164.1, 153.6, 148.9, 147.9, 147.7, 136.0, 134.5, 129.6, 129.3, 124.7,
121.2, 120.1, 119.8, 119.6, 114.7, 113.3, 61.1, 56.9, 56.0, 41.8, 34.7, 25.9, 18.3; HRMS (ESI)
m/z calcd. for C26H29N2O6S 497.1746 [M + H]+; found: 497.1745.
4.1.2.28 (1E, 4E)-6-(1-(2-Fluorobenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-
thoquinone dioxime (9q)
o
1
Light yellow solid, yield 74%, m.p.: 195–198 C; H NMR (400 MHz, DMSO-d6): δ
12.18 (s, 2H, 2 × (N=OH)), 7.84 – 7.79 (m, 1H, ArH), 7.69 (dd, J = 8.0, 3.6 Hz, 2H, ArH),
7.42 – 7.34 (m, 4H, QuinH and ArH), 7.22 (s, 1H, ArH), 5.28 (t, J = 7.4 Hz, 1H, CH2CH=C),
5.10 (t, J = 6.8 Hz, 1H, ArCHCH2), 3.83 (s, 3H, OCH3), 3.66 (s, 3H, OCH3), 2.84 – 2.73 (m,
2H, CHCH2CH=C), 1.64 (s, 3H, =C-CH3), 1.62 (s, 3H, =C-CH3); 13C NMR (100 MHz,
DMSO-d6): δ 187.7, 160.8, 158.3, 153.7, 148.9, 147.6, 135.5, 134.7, 129.8, 125.5, 124.7,
121.0, 120.1, 119.9, 119.6, 117.6, 117.4, 113.3, 61.1, 57.0, 42.4, 34.4, 25.9, 18.3; HRMS (ESI)
m/z calcd. for C25H26FN2O5S 485.1546 [M + H]+; found: 485.1544.

ACCEPTED MANUSCRIPT
4.1.2.29 (1E, 4E)-6-(1-(3-Fluorobenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-
thoquinone dioxime (9r)
o
1
Light yellow solid, yield 78%, m.p.: 190–192 C; H NMR (400 MHz, DMSO-d6): δ
12.11 (s, 2H, 2 × (N=OH)), 7.80 (d, J = 7.6 Hz, 1H, ArH), 7.67 (d, J = 8.0 Hz, 1H, ArH), 7.63
– 7.55 (m, 2H, ArH), 7.39 (s, 2H, QuinH), 7.22 (s, 1H, ArH), 5.27 (t, J = 7.4 Hz, 1H,
CH2CH=C), 5.08 (t, J = 7.4 Hz, 1H, ArCHCH2), 3.82 (s, 3H, OCH3), 3.65 (s, 3H, OCH3),
2.85 – 2.79 (m, 1H, 1/2 × (CHCH2CH=C)), 2.77 – 2.72 (m, 1H, 1/2 × (CHCH2CH=C)), 1.64
13
(s, 3H, =C-CH3), 1.60 (s, 3H, =C-CH3); C NMR (100 MHz, DMSO-d6): δ 189.7, 163.8,
161.3, 153.7, 148.9, 147.8, 147.6, 138.7, 135.5, 134.8, 131.9, 124.7, 123.7, 120.9, 120.2,
119.6, 113.9, 113.6, 113.3, 61.1, 57.0, 42.4, 34.5, 25.9, 18.3; HRMS (ESI) m/z calcd. for
C25H26FN2O5S 485.1546 [M + H]+; found: 485.1540.
4.1.2.30 (1E, 4E)-6-(1-(4-Fluorobenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-
thoquinone dioxime (9s)
o
1
Light yellow solid, yield 85%, m.p.: 179–183 C; H NMR (400 MHz, DMSO-d6): δ
12.12 (s, 2H, 2 × (N=OH)), 8.02 (dd, J = 8.0, 4.0 Hz, 2H, ArH), 7.43 – 7.36 (m, 4H, QuinH
and ArH), 7.24 (s, 1H, ArH), 5.28 (t, J = 7.6 Hz, 1H, CH2CH=C), 5.09 (t, J = 7.0 Hz, 1H,
ArCHCH2), 3.84 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 2.88 – 2.71 (m, 2H, CHCH2CH=C),
1.65 (s, 3H, =C-CH3), 1.61 (s, 3H, =C-CH3); 13C NMR (100 MHz, DMSO-d6): δ 189.3, 167.0,
164.5, 153.6, 148.9, 147.8, 147.6, 135.7, 134.7, 133.2, 130.3, 130.2, 124.7, 121.0, 120.1,
119.9, 119.6, 116.7, 116.5, 113.3, 61.1, 56.9, 42.2, 34.5, 25.9, 18.2; HRMS (ESI) m/z calcd.
for C25H26FN2O5S 485.1546 [M + H]+; found: 485.1539.
4.1.2.31 (1E, 4E)-6-(1-(2-Chlorobenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-
thoquinone dioxime (9t)
o
1
Light yellow solid, yield 81%, m.p.: 199–202 C; H NMR (400 MHz, DMSO-d6): δ
12.13 (s, 2H, 2 × (N=OH)), 7.67 (d, J = 8.0 Hz, 1H, ArH), 7.62 – 7.56 (m, 2H, ArH), 7.50 –
7.45 (m, 1H, ArH), 7.41 (s, 2H, QuinH), 7.21 (s, 1H, ArH), 5.28 (t, J = 7.6 Hz, 1H,
CH2CH=C), 5.12 (t, J = 7.0 Hz, 1H, ArCHCH2), 3.83 (s, 3H, OCH3), 3.68 (s, 3H, OCH3),
13
2.88 – 2.74 (m, 2H, CHCH2CH=C), 1.65 (s, 3H, =C-CH3), 1.63 (s, 3H, =C-CH3); C NMR
(100 MHz, DMSO-d6): δ 191.0, 153.7, 148.8, 147.7, 137.4, 135.4, 134.8, 133.4, 131.1, 129.5,
129.3, 128.1, 124.7, 120.9, 120.2, 119.6, 113.2, 61.2, 57.0, 43.1, 34.6, 26.0, 18.3; HRMS (ESI)

ACCEPTED MANUSCRIPT
m/z calcd. for C25H26ClN2O5S 501.1251 [M + H]+; found: 501.1248.
4.1.2.32 (1E, 4E)-6-(1-(3-Chlorobenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-
thoquinone dioxime (9u)
o
1
Light yellow solid, yield 74%, m.p.: 196–198 C; H NMR (400 MHz, DMSO-d6): δ
12.03 (s, 2H, 2 × (N=OH)), 7.82 (d, J = 7.6 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 7.68 (d, J = 8.0
Hz, 1H, ArH), 7.50 (t, J = 7.8 Hz, 1H, ArH), 7.32 (s, 2H, QuinH), 7.15 (s, 1H, ArH), 5.20 (t, J
= 7.6 Hz, 1H, CH2CH=C), 5.01 (t, J = 7.0 Hz, 1H, ArCHCH2), 3.76 (s, 3H, OCH3), 3.58 (s,
3H, OCH3), 2.79 – 2.63 (m, 2H, CHCH2CH=C), 1.56 (s, 3H, =C-CH3), 1.52 (s, 3H, =C-CH3);
13C NMR (100 MHz, DMSO-d6): δ 189.6, 153.6, 148.8, 147.8, 147.6, 138.3, 135.4, 134.7,
134.4, 134.1, 131.6, 126.7, 126.1, 124.7, 120.9, 120.1, 119.9, 119.6, 113.2, 61.1, 56.9, 42.4,
34.4, 25.9, 18.2; HRMS (ESI) m/z calcd. for C25H26ClN2O5S 501.1251 [M + H]+; found:
501.1239.
4.1.2.33 (1E, 4E)-6-(1-(4-Chlorobenzoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naph-
thoquinone dioxime (9v)
o
1
Light yellow solid, yield 79%, m.p.: 187–189 C; H NMR (400 MHz, DMSO-d6): δ
12.13 (s, 2H, 2 × (N=OH)), 7.95 (d, J = 8.0 Hz, 2H, ArH), 7.62 (d, J = 8.0 Hz, 2H, ArH), 7.41
(s, 2H, QuinH), 7.23 (s, 1H, ArH), 5.28 (t, J = 7.0 Hz, 1H, CH2CH=C), 5.09 (t, J = 7.0 Hz, 1H,
ArCHCH2), 3.84 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 2.87 – 2.70 (m, 2H, CHCH2CH=C),
1.64 (s, 3H, =C-CH3), 1.61 (s, 3H, =C-CH3); 13C NMR (100 MHz, DMSO-d6): δ 189.7, 153.7,
148.9, 147.9, 147.7, 139.3, 135.6, 135.3, 134.7, 129.7, 129.1, 124.7, 121.0, 120.1, 119.9,
119.6, 113.3, 61.1, 57.0, 42.4, 34.5, 25.9, 18.3; HRMS (ESI) m/z calcd. for C25H26ClN2O5S
501.1251 [M + H]+; found: 501.1246.
4.1.2.34 (1E, 4E)-6-(1-(2-Furoylthio)-4-methylpent-3-enyl)-5,8-dimethoxy-1,4-naphthoquino-
ne dioxime (9w)
o
1
Light yellow solid, yield 81%, m.p.: 176–179 C; H NMR (400 MHz, DMSO-d6): δ
12.04 (s, 2H, 2 × (N=OH)), 7.98 (d, J = 3.6 Hz, 1H, OCH=CH), 7.35 (d, J = 3.6 Hz, 1H,
C=CH-CH), 7.34 (s, 2H, QuinH), 7.15 (s, 1H, ArH), 6.70 (t, J = 4.0 Hz, 1H, CH=CH=CH),
5.19 (t, J = 7.6 Hz, 1H, CH2CH=C), 5.01 (t, J = 6.8 Hz, 1H, ArCHCH2), 3.77 (s, 3H, OCH3),
3.58 (s, 3H, OCH3), 2.79 – 2.71 (m, 1H, 1/2 × (CHCH2CH=C)), 2.70 – 2.62 (m, 1H, 1/2 ×
(CHCH2CH=C)), 1.57 (s, 3H, =C-CH3), 1.55 (s, 3H, =C-CH3); 13C NMR (100 MHz,