New Spisulosine Derivative promotes robust autophagic response to cancer cells

Article abstract of DOI:10.1016/j.ejmech.2019.112011

Therapy resistance by evasion of apoptosis is one of the hallmarks of human cancer. Therefore, restoration of cell death by non-apoptotic mechanisms is critical to successfully overcome therapy resistance in cancer. By rational drug design approach, here we try to provide evidence that subtle changes in the chemical structure of spisulosine completely switched its cytotoxic function from apoptosis to autophagy. Our most potent molecule (26b) in a series of 16 synthesized derivatives showed robust autophagic cell death in diverse cancer cells sparing normal counterpart. Compound 26b mediated lethal autophagy induction was confirmed by formation of characteristic autophagic vacuoles, LC3 puncta formation, upregulation of signature autophagy markers like Beclin and Atg family proteins. Altogether, we have detected novel autophagy inducer small molecule which can be tested further for drug discovery research.

Full text of DOI:10.1016/j.ejmech.2019.112011

Journal Pre-proof
New Spisulosine Derivative promotes robust autophagic response to cancer cells
Asha Ganesher, Priyank Chaturvedi, Rohit Sahai, Sanjeev Meena, Kalyan Mitra,
Dipak Datta, Gautam Panda
PII:
S0223-5234(19)31168-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.112011
EJMECH 112011
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 June 2019
Revised Date: 2 December 2019
Accepted Date: 24 December 2019
Please cite this article as: A. Ganesher, P. Chaturvedi, R. Sahai, S. Meena, K. Mitra, D. Datta, G.
Panda, New Spisulosine Derivative promotes robust autophagic response to cancer cells, European
Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.112011.
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Graphic Abstract
New Spisulosine Derivative Promotes Robust Autophagic Response to Cancer Cells
Asha Ganesher, Priyank Chaturvedi, Rohit Sahai, Sanjeev Meena, Kalyan Mitra, Dipak Datta,
Gautam Panda
Compound 26b promotes Autophagic cell death

New Spisulosine Derivative Promotes Robust Autophagic Response
to Cancer Cells
,a
,b
Asha Ganesher , Priyank Chaturvedi , Rohit Sahaic, Sanjeev Meenab, Kalyan Mitrac, Dipak
Datta*,b, Gautam Panda*,a
a
Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Sitapur
Road, Jankipuram Extension, Lucknow 226031, UP, India
b
Division of Cancer Biology, CSIR-Central Drug Research Institute, Sitapur Road, Jankipuram
Extension, Lucknow 226031, UP, India
c
Electron Microscopy Unit, Sophisticated Analytical Instrument Facility, CSIR-Central Drug
Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226 031, UP, India
equal contribution
Corresponding author
*Gautam Panda, Phone: 915222772450, ext 4659, Fax: 915222771941;
E-mail: gautam.panda@gmail.com; gautam_panda@cdri.res.in
*Dipak Datta, Phone 915222772450, ext 4347, Fax: 915222771941;
E-mail: dipak.datta@cdri.res.in
KEYWORDS: Sphingoid, chemotherapy, autophagy, resistance, structure-activity relationship
ABBREVIATIONS
br s, broad signal; rt, room temperature; mp, melting point; THF, tetrahydrofuran; DCM,
dichloromethane; MeOH, methanol; K2CO3, potassium carbonate; NaHCO3, sodium
bicarbonate; MOM-Cl, chloromethyl methyl ether; MsCl, methanesulfonyl chloride; Et3N,
triethylamine;
Boc
anhydride,
di-tert-butyl
dicarbonate;
LiAlH4,
lithium
alumininumtetrahydride; TBDPS-Cl, tert-Butyldiphenylchlorosilane
ABSTRACT
Therapy resistance by evasion of apoptosis could be one of the hallmarks of human cancer.
Therefore, restoration of cell death by non-apoptotic mechanisms is critical to successfully
overcome therapy resistance in cancer. By rational drug design approach, here we try to provide
evidence that subtle changes in the chemical structure of spisulosine completely switched its
cytotoxic function from apoptosis to autophagy. Our most potent molecule (26b) in a series of 16
synthesized derivatives showed robust autophagic cell death in diverse cancer cells sparing
normal counterpart. Compound 26b mediated lethal autophagy induction was confirmed by
formation of characteristic autophagic vacuoles, LC3 puncta formation, upregulation of signature
autophagy markers like Beclin and ATG family proteins. Altogether, we have detected novel
autophagy inducer small molecule which can be tested further for drug discovery research.
1

1. INTRODUCTION
Cancer is one of the most serious life-threatening diseases in the world. During past several
years, huge efforts have been made by researchers but the search of effective clinical approaches
for the treatment of cancer is still a tough challenge. Chemotherapy using anticancer agents is
another valuable option for the cancer treatment apart from surgery, immunotherapy, and
radiotherapy.1In the last few years, it is now very common in the developed countries.2 So, the
development of novel anticancer agents is a highly active research field and has achieved
considerable attention from chemists.3 Presently, most of the drugs in clinical trials for cancer
treatment are natural products or pharmacophores derived from natural product sowing to their
fewer side effects, thus the impact of natural products upon anticancer drug discovery and design
is very inspiring.4,5 However, in the initial phase of these active natural or synthetic compounds,
poor solubility is one of the major issues.6 The poor prognosis of colon cancer and poor
sensitivity to current therapeutics, associated with resistant to apoptosis, urge the search of new
drugs which induce cancer cell death by some other mechanisms.7
Long-chain α-amino-alcohols i.e. sphingoid-type bases are considered as the principal backbone
of the more complex sphingolipids and ceramides8,9,10(Fig1). Ceramides show its mechanism of
action through cellular signaling and activating various protein kinase cascades whereas
sphingolipids show diverse biological activities such as antitumor,11,12,13 immunostimulatory, and
immunosuppressive,14neuronal proliferation15and protein kinase activity variation. Sphingosine
is also a sphingoid base, which is a derivative of sphinganine 2.16 Over the past decade, 1-
deoxysphingoid bases have received increased attention from synthetic chemists due to their
important biological activity.17 Among them, spisulosine 3 has received the most attention.
Fig. 1 Structures of several natural sphingoid bases
2

(2S,3R)-2-Amino-3-octadecanol (spisulosine or ES-285) is a marine derived bioactive compound
isolated from the North Arctic clam Spisula polynyma18 by Rinehart et al. This compound is
known for its biological activity to inhibit cell proliferation with an IC50 of 1µM in the prostate
tumor PC-3 and LNCaP cell lines19and is responsible for loss of actin stress fibres.20The
mechanism of action of spisulosine is still hypothetical. Ceramide signaling and GTP binding
protein (RHO protein) regulating actin stress fibers have been proposed as a potential molecular
target.21Although,spisulosine was primarily developed as a novel anticancer agent but due to its
poor clinical outcomes it was discontinued from phase I in 2008.22,23ES-285 showed a tolerable
safety profile at doses up to 128 mg/m2. In a number of clinical studies of various compounds of
natural origin and especially with some marine agents, drug-related neurotoxicity has been a
common problem.24,25,26 In the phase I program of ES-285, the neurological disorders like
dizziness, headache, sensory or motor neuropathy, neuropathic pain, aphasia, and decreased level
of consciousness was detected. One patient died after the first administration of the ES-285 when
treated at 200 mg/m2for 24-h i.v. infusion following drug related central neurotoxicity. Other
toxicities included phlebitis, nausea, fatigue, and fever. The pharmacokinetic studies revealed
high volume of distribution and long elimination half-life, which led to discontinuation of this
compound. Due to an unfavorable risk/profit balance and absence of clinically significant
antitumor efficacy in the whole clinical program, recruitment of patients to spisulosine clinical
trials was discontinued. The seriousness of the neurological toxicity experienced by one patient
supported this decision.27
However, this compound is structurally similar to other related 1-deoxysphingoids with
remarkable cytotoxic properties, such as obscuraminols,28 clavaminols,29crucigasterins,30 and
xestoaminols,31 (Fig.1). This close structural relationship makes this type of compound as a
prominent lead for anticancer oriented drug discovery programs.In literature, various methods
are reported for the total synthesis of this natural product,32,33,34,35,36,37,38,39,40,41 in which chiral
amino acid, carbohydrate, garner aldehyde and various achiral substrates like palmityl alcohol,
palmitaldehyde, pentadec-1-yne etc have been used as an starting material (Fig. 2).
3

Fig. 2 Known approaches for total synthesis of spisulosine (ES-285)
However, only few reports of analogs of spisulosine are present in the literature. So far, only
Delgado et al.,42Bittman et al.43 and Dauban et al.44 have synthesized spisulosine analogs (10-19)
and evaluated for biological activity in order to evaluate the ceramides synthase activity in whole
cells (Fig.3).45Bittman et al. found that analogue 10 shows IC50= 27.8±3.2 µM against
sphingosine kinase (SphK1 isoform) and analogue11 was found to be a nonselective SphKs
inhibitor.Delgado et al. have synthesized 3 and its analogues such as stereoisomers 12, 13 and
14, and dehydrospisulosines15, 16, 17 and 18 in order to analyse the ceramides synthase activity
in whole cells (Fig.3). Only three compounds (spisulosine 3, 3-epimer 14 and 4,5-
dehydrospisulosine 18) were found to be most active for further screening. Recently, Dauban et
al. have synthesized spisulosine 3 and its 3-fluoro derivative 19and screened for biological
evaluation in three malignant cells (KB, HCT-116, HL-60). As expected, only compound 3
exhibited cytotoxicity (IC50 in the 100 nM range).
4

Fig. 3 Several analogs of spisulosine 3
Although much synthetic and biological effort in the field of 1-deoxysphingoid bases and their
related compounds have been done, but many challenges in this research still remain. All of
these natural compounds are an interesting source of inspiration for both the organic community
and biomedical research, and further intensive studies in this area could provide a new insight
into the design and mechanism of actionof the sphingolipid related anticancer agents. Inspired
from these analogs and their biological activity, we have synthesized a new derivative of
spisulosine which induces cancer cell death by autophagy. Although the role of autophagy in
cancer is very complex, various pharmacological agents with different antineoplastic properties
have been shown to induce autophagic activity resulting in massive death of cells in some cancer
types.For example in renal cell, carcinomas are refractory to standard therapies, but are sensitive
for autophagy induced cell death.46 Apoptosis resistance is one of the major causes of
chemoresistance, and an important challenge in the treatment of cancer is the development of
therapies that overcome chemoresistance. Thus activation of autophagy in apoptosis resistant
cancer could potentially provide a way to induce cell death and impede malignant growth.
Rationale of the design
5

Fig. 4 Approach towards designing of spisulosine and sphinganine analogs.
The simple 1,2-amino alcohols have been observed to show a rare type of bioactivity such as
anti-proliferative and cytotoxic activities along with their ability to influence the sphingolipids
biosynthesis and metabolism. Because of their wide applications in biological field, some of
these sphingosine related natural products have been selected as lead molecule for the design of
new anticancer agents.47
Structural analysis of all the sphingoid type bases revealed that 1,2 amino alcohol (the body
region) is highly preserved. From literature survey, we hypothesized that the poor
pharmacokinetic profile perhaps arises from the strong intra-molecular hydrogen bonding
between two adjacent amino and hydroxyl functionalities present in spisulosine skeleton which
may be responsible for its long elimination half-life. Thus, the free amino and hydroxyl group
were protected to observe the effect of substitution on water solubility as well as activity (Fig.4).
Further, the head region of spisulosine was modified with substituted phenyl groups, cyclic and
acyclic tertiary amines in order to see the effect of hydrophobicity/hydrophilicity on their
activity. Learned from the earlier investigations34-37, the tail region was kept intact with 14
carbon atoms.
2. RESULTS AND DISCUSSION
6

Chemistry
The designed spisulosine derivatives 25 and 26b were synthesized according to the procedure
reported in scheme 1. Naturally abundant L-tyrosine amino acid was taken as starting material.
Firstly, L-tyrosine methyl ester hydrochloride 20 was protected in presence of Boc anhydride to
furnish Boc protected tyrosine methyl ester 21 in good yield (86%). In the next step, the free OH
of compound 21 was methylated in presence of methyl iodide by using K2CO3 as a base in
acetone to provide compound 22 in very good yield (88%), which was subjected to
LiAlH4mediated reduction of the ester group to afford alcohol 23 in excellent yield (93%). Dess-
Martin oxidation in DCM-H2O afforded aldehyde 24 within 1h whose immediate treatment with
tetradecylmagnesium chloride Grignard reagentafforded the syn-diastereomer 25 in good yield
(73% over two steps) according to the known chelation controlled addition of Grignard reagents
to the Boc protected amino aldehydes to produce the corresponding β-amino alcohol with high
syn-selectivity.48 The formation of the syn adduct 25 was explained with the Cram model having
a chelation-controlled transition state (Fig.5).49 Finally, removal of the protecting group under
acidic condition (4N HCl in dioxane) afforded diastereomerically pure (2S,3S)-2-amino-1-(4-
25
methoxyphenyl)octan-3-ol 26b {[α]D -10.0 (c 0.05, MeOH) in 65% yield but equal amount of
demethylated product 26a was also observed. To reduce the minor product 26a, Boc
deprotection with Dowex® 50Wx8 resin in MeOH was done and 26b as the major product was
formed and only 5% of minor 26a was observed.
Scheme 1. Reagents: a) Boc anhydride, NaHCO3, MeOH, 0 °C- RT, 4 h, 86 % b) CH3I, K2CO3,
acetone, 0 °C- RT, 4-5 h, 88 % c) LiAlH4, dry THF, 0 °C- RT, 1 h, 93 % d) Dess-Martin
Periodinane, DCM-H2O, 0 °C- RT, 1 h e) C14H29MgCl, dry THF, 0 °C- RT, 4-5 h, 73 % over
7

two steps f) 4N HCl in dioxane, RT, 0.5 h, 65 % (50% conversion) or Dowex® 50Wx8 resin,
MeOH, 50 °C, 5 h, 85 % ( 95 % conversion).
O
O
M
O
O
H
O
Nu
O
NH
NHBoc
N
R
H
H
R
CHO
R
(Si)
Nu
H
H
(Re)
less stable
more stable
anti
syn
NHBoc
Nu
NHBoc
Nu
R
R
OH
minor, 5%
OH
major, 95%
Fig. 5 Syn-productformation bynucleophilic additions via chelation-controlled Cram model.
Another explanation of this syn-stereochemistry was explained by Ichihashi and Mori in 200350
by converting diastereomeric mixture of Boc protected amino alcohol to the corresponding
oxazolidines. By conducting NOE experiment on this mixture of oxazolidines exposed that syn-
adduct was the major isomer.
Spisulosine derivatives 30, 32, 33, 34, 36a-f, 37a-d, were synthesized according to the scheme 2.
Almost same procedure was used as mentioned in scheme 1. Instead of using L-tyrosine, L-
serine amino acid was used as a starting material in this scheme. Firstly, L-serine methyl ester
hydrochloride 27was protected by using Boc anhydride and NaHCO3 as a base to provide Boc
protected serine methyl ester 28 in excellent yields (94%). The free -OH group of compound 28
was then protected with TBDPS-Cl in presence of imidazole to give compound 29 in very good
yield ( 89%), which was then subjected to LiAlH4 mediated reduction of ester to afford alcohol
30 in good yield ( 88%). Further, the alcohol 30 was oxidized through Dess-Martin oxidation in
DCM-H2O to furnish aldehyde 31, which was then subjected to Grignard reaction by using
Grignard reagent tetradecylmagnesium chloride in THF solvent to provide long chain syn-
diastereomer 32 in good yield (81% over two steps) according to the above chelation controlled
Cram model (Fig.5). Compound 32 was then reacted with MOM-Cl by using DIPEA as a base to
give compound 33in 79 % yield which was further reacted with TBAF to remove the TBDPS
group to furnish compound 34 with good yield (78%), the free –OH of compound 34 was then
reacted with MsCl by using Et3N as a base to make it a good living group (compound 35).
Substitution reaction was done by using different secondary amines in acetonitrile under reflux
condition on compound 35 to furnish compounds 36a-f in good yields (64-72%) with retention
8

of syn-stereochemistry. In the last step, the MOM group was removed by using ethane dithiol in
presence of BF3.OEt2 in dry DCM to afford compounds 37a-d in good yields (69-72%).
OH
OH
OTBDPS
COOMe
a
b
c
ClH.H2N
COOMe
COOMe
BocHN
BocHN
27
28
29
OTBDPS
OH
OTBDPS
OH
TBDPSO
e
d
f
H
O
BocHN
31
10
BocHN
NHBoc
32
30
OMOM
g
OMOM
HO
MsO
OMOM
h
TBDPSO
10
10
10
NHBoc
34
NHBoc
35
NHBoc
33
OMOM
R
OH
R
i
j
10
10
NHBoc
36a-f
NHBoc
37a-d
36a, R= N(CH2CH3)2
36b, R= N((CH)(CH3)2)2
36c, R= N((CH2)3(CH3))2
37a, R= N(CH2CH3)2
37b, R= N((CH)(CH3)2)2
37c, R= N((CH2)3(CH3))2
N
36d, R=
36e, R=
N
37d, R=
S
S
N
N
36f, R=
N
Scheme 2. Reagents: a) Boc anhydride, NaHCO3, MeOH, 0 °C- RT, 4 h, 94 % b) TBDPS-Cl,
imidazole, dry DCM, 0 °C- RT, 2 h, 89 % c) LiAlH4, dry THF, 0 °C- RT, 1h, 88 % d) Dess-
Martin Periodinane, DCM-H2O, 0 °C- RT, 1 h e) C14H29MgCl, dry THF, 0 °C- RT, 4-5 h, 81 %
over two steps f) MOM-Cl, DIPEA, dry DCM, 0 °C- RT, 2-3 h, 79 % g) TBAF, dry THF, 0 °C-
RT, 4-5 h, 78 % h) MsCl, Et3N, dry DCM, 0 °C- RT, 4-5 h, 84 % i) RH, CH3CN, 0 °C- reflux, 5-
6 h, 64-72 % j) Ethane dithiol, BF3.OEt2, dry DCM, 0 °C- RT, 2 h, 69- 72 %.
9

Biological Evaluation:
Novel spisulosine analogs are cytotoxic against cancer cells
We screened a series of 16 compounds for their cytotoxic activity against six different cancer
cell lines representing three cancer types by using well established SRB assay as described in
method section. Representative cell lines from different cancer types are DLD-1, HT-29
(Colorectal adenocarcinoma), MCF-7, MDA-MB231 (Breast adenocarcinoma), A549, NCI-
H358 (Lung carcinoma). Detailed results of these investigations in terms of percentage
inhibitions of cell growth at 10 µM of each compound are given in Table 1. Here, we observed
compound 26b was found to be potentially active against all the tested cell lines at a 10 µM dose,
having maximum effect in colon cancer cell lines (more than 95% cytotoxicity in both the cell
lines). Next, we determined its IC50 in a panel of colon cancer cell lines as mentioned in the
bottom panel which was turned out to be less than 5 µM in all the colon cancer cells (Fig.6).
10

Table 1: Percentage (%) cell growth inhibition at 10µM dose in diverse cancer cell lines
Breast
Colon
Lung
Sr.
No.
Compound
Code
Structure
MCF- MDA- DLD- HT- A549 NCI-
7
MB-
231
1
29
H358
1
2
8.77
-16.50 27.95 -7.41 23.41 6.37
25
32
2.79
-37.45 10.42 -14.40 14.57 3.11
3
4
89.28
96.84
97.30 97.66 94.85 90.40
26b
33
-7.95 -27.98 -14.62 -18.76 15.13 -2.89
5
6
10.15
25.25
-5.67
0.84
54.84 22.02 25.91 18.43
46.23 -3.21 27.47 14.95
34
36a
7
8
26.91
27.06
30.98
22.26
53.90 19.92 22.63 26.58
56.05 14.98 42.23 25.12
36b
36c
9
27.34
23.99
9.84
4.73
43.70 14.19 18.98 15.22
36e
36d
10
58.55
8.57 29.92 22.68
11

11
28.01
24.00
31.95 19.34 18.25 18.86
36f
OTBDPS
12
13
28.26
25.28
32.29
-1.17
73.50 46.25 25.45 53.43
34.83 11.62 22.93 11.26
30
OH
BocHN
37b
14
18.71
21.54
-1.45
-9.61
14.73 15.30 18.52 8.37
37a
15
16
23.42
9.83 29.97 9.40
37c
37d
30.25
88.50
6.84
32.37 12.04 31.96 10.81
76.68 78.40 80.25 77.46
Doxorubicin
(10µM)
80.16
Structure-activity-relationship studies
Based on the biological data reported in Table 1, many structure activity relationships (SAR)
could be deduced. According to the results, we found that the compound 26b with 4-
methoxyphenyl in the head region and free amine and hydroxyl functionality in the body region
was found to be more potent and showed better inhibitory activities against all the tested cell
lines at a 10 µM dose, showing maximum effect in colon cancer cell lines (more than 95%
cytotoxicity in both the cell lines) whereas its protected analog 25 (Boc protected amine) was 3
to 100-fold less potent than compound 26b.
To further optimize the lead compound 26b, we performed a SAR study on the head and body
region by introducing different tertiary amines in the head region and protecting the free amine
and hydroxyl group of the body region (compound no- 36a to 36f). As shown in Table 1, most
12

of the compounds showed only moderate activity in DLD-1 cell line. Further exploration of SAR
at the body region was conducted by protecting only the amine group and keeping the hydroxyl
group free (compound no- 37a to 37d) but the results were not satisfactory, there was
tremendous decrease in activity. Hence, from the SAR studies it was clear that the free amine
and hydroxyl group was crucial for the activity. With this structure-activity-relationship analysis,
analogue 26b was selected for further evaluation.
13

Fig. 6 Novel Spisulosine analogs are cytotoxic against colon cancer cells. Different human cancer
cells (MCF-7, MDA-MB-231, A549, NCI-H358, DLD-1, and HT-29) were treated with 10ꢀµM of different
Spisulosine derivatives for 48ꢀhours and cytotoxicity was measured by SRB assay as described in Materials
and Methods. Percent growth inhibitions were tabulated (Top Panel). Various human colon cancer cells
(COLO-205, LOVO, HT-29, DLD-1, SW48, and SW-620) and normal human colon fibroblast (CCD-
18Co) cells were treated with varying concentrations of compound 26b for 48 h and the half maximal
proliferation inhibitory concentration (IC50) values were determined (Bottom Panel) by SRB assay.
Columns, average of triplicate readings of samples; error bars, ± S.D.
Compound 26b promotes non apoptotic cell death
Potent cytotoxic action of compound 26b prompted us to determine its primary mode of action.
To our great surprise, we observed that compound 26b did not pose its cytotoxic effects through
classical apoptosis (Fig. 7A-D). It did not result in neither generation of early apoptotic cells as
detected by Annexin-V-PI staining nor cleavage of hallmark apoptotic features like PARP
protein. Moreover, it promotes a robust increase in cellular granularity through vacuole
formation (Fig. 7B). In contrast, parent spisulosine treatment has been shown to increase
Annexin-V positive cells as well as PARP cleavage indicating an active apoptotic process.
14

Fig.7 Compound 26b promotes non-apoptotic cell death. Panel A-D, DLD-1 cells were treated with
either vehicle or Compound 26b or Spisulosine at 7.5 µM for 18 hours and analyzed for early apoptosis
(Annexin-V staining by FACS) and Cleaved PARP expression (Western Blot). Representative photo
micrographs were shown in panel A. Cell size (FSC) and cellular Granularity (SSC) were shown by
FACS in panel B. In panel C, representative FACS plots show Annexin-V-FITC and PI stained cells.
Immunoblot analysis (panel D) shows the expression of cleaved PARP (Apoptotic marker protein)
following protein isolation of vehicle and treated cells.
Compound 26b promotes Autophagic cell death
As we found, the cytotoxic cell death induced by compound 26b was not due to apoptosis, and
also observed that compound 26b treatment promotes intracytoplasmic vacuole accumulation in
colon cancer (DLD-1) cells, which is characteristic feature of cells undergoing autophagy. To
confirm the formation of autophagic vesicles, we stained cells with a fluorescent compound
MDC (monodansylcadaverine) which is an autophagy marker and act as lysomotrophic agent
and labels the acidic compartments of fused lysosomes51,52. Under confocal microscopy analysis,
15

we observed vacuoles were positively stained by MDC and appeared as distinct dot like
structures distributing in cytoplasm of DLD-1 cells treated by compound 26b; the fluorescent
density of MDC-labeled particles in DLD-1 cells were higher in compound 26b treated group
than in control group (Fig. 8A) suggesting autophagy induction upon compound treatment. LC3
lipidation is a classical biomarker of cellular autophagy levels. During autophagy, the 16-kDa
cytosolic (LC3-I) form is post-translationally modified to form 14kDa (LC3-II) and translocated
to autophagosome. The western blot analysis results (Fig. 8B) shown an increase in conversion
of LC3B-I to LC3B-II. Further, we have confirmed our observation by determining the LC-3B-II
puncta formation following compound treatments at different doses (Fig. 8C).
Fig.8 Compound 26b promotes Autophagic cell death. Panel A-B, DLD-1 cells were treated
with either vehicle or compound 26b at 7.5 µM for 18 hours. Cells were stained with autophagy
marker MDC (monodancycadaverin) and analyzed under confocal microscope (panel A). Cells
were harvested for protein isolation and subjected to immunoblot for LC-3 and GAPDH
expression (panel B). DLD-1 cells were treated with either vehicle or compound 26b for 6 hours
16

and subjected to confocal microscopy for determination of LC-3 puncta formation (panel C).
DLD-1 cells were treated with either vehicle or compound 26b at 10 µM for indicated hours.
Cells were harvested for protein isolation and subjected to immunoblot for Beclin and Atg
protein expression (panel D)
Moreover, compound 26b robustly induces the expression of classical autophagy markers like
Beclin-1, Atg3, Atg5 and Atg7, compared to control at multiple time points (Fig. 8D).
To further confirm our previous observations, we performed TEM analysis of control and treated
cells. As observed in Fig. 9, compound 26b induced numerous classical autophagic vacuoles
characterized by the presence of double membrane vesicles with cargo. Compared to control
cells, no mitochondrial or nuclear damage were observed in the treated cells, suggesting absence
of apoptosis in response to compound treatment.
Fig. 9 Compound 26b induces autophagic vacuole formation. Representative transmission
electron micrographs of control (A-C) and treated (D-F) DLD-1 colorectal adenocarcinoma
cells. Untreated cells display normal ultrastructure of organelles like mitochondria, rough ER
and nucleus (B, C). Autophagic vacuoles (arrows) were observed in the treated (5 µM for 6
17
hours) cells. No structural alterations were observed in the mitochondria in treated cells
indicating autophagy selective responses.

To test whether compound 26b induced autophagy linked with cellular cytotoxicity, we
pretreated our cells with bafilomycinA1, a specific autophagy inhibitor, and observed the
To test whether compound 26b induced autophagy linked with cellular cytotoxicity, we
pretreated our cells with Bafilomycin-A1, a specific autophagy inhibitor, and observed the
attenuation of compound 26b induced autophagic vacuole formation and subsequent cell death
compared with only compound 26b treated cells. Interestingly, pretreatment of Bafilomycin in
presence of parent compound spisulosine in DLD-1 cells did not inhibit cytotoxic cell death
indicative of non autophagic cell death (Fig. 10A and 10B).
Based on this data, we concluded that the major difference between parent compound spisulosine
and our potent compound 26b was para-methoxyphenyl part which may be responsible for the
autophagic cell death because the parent compound was going through classical apoptosis and
our compound 26b induced cell death via vacuole formation.
18

Fig. 10 Compound 26b promotes autophagic cell death. DLD1 cells were treated with either
vehicle or compound 26b (7.5 µM) or autophagy inhibitor Bafilomycin A1 (100nm) plus
compound 26b (7.5 µM) for 24 hours and analyzed under phase-contrast microscope (panel A).
Cellular cytotoxicity was performed by crystal violet staining in control and treated (48hours)
cells and representative photomicrographs were shown (panel B).
3. CONCLUSION
We have synthesized a series of spisulosine analogs based on a rational design taking inputs
from various sphingoid type bases having anticancer effects. The aim was to create a novel
library with better pharmacokinetic profile and to determine its mechanism of action. During our
search toward its cytotoxic mode of action, we surprisingly discovered that unlike parent
spisulosine, compound 26b promoted robust autophagic cell death to cancer cells instead of
classical apoptosis. Compound 26b treatment resulted in massive vacuole formation which was
found to be positive for autophagy marker expression like LC3-II puncta formation, upregulation
of Atg proteins etc. Biochemically, it induced classical autophagy markers but not promoted
hallmark apoptotic features like cleavage of PARP or caspases. Cytotoxic effect of compound
26b can be effectively rescued by the autophagy inhibitor but not in case of parent spisulosine.
Overall, we discovered a new small molecule that markedly induces autophagic cell death to
various cancer cells.
4. EXPERIMENTAL PROTOCOLS
Materials and methods
All commercially available starting materials and reagents were used without further
purification. Organic solvents were dried by standard methods. All dry reactions were carried out
13
under argon or nitrogen atmosphere. All the compounds were characterized by 1H, C, ESI-MS
and HRMS. All reactions were monitored by TLC using 2.5x5 cm plates coated with 0.25 mm
thickness of silica gel. Visualization was done by using UV light and iodine vapors. Purification
was done by silica gel (100-200 mess) column chromatography by using freshly distilled
solvents. NMR spectra were recorded on BrukerAvance DPX 200FT, Bruker Robotics, Bruker
1
13
DRX 400, 100 and 125 (400 MHz for H and 100, 125 MHz for C) using CDCl3 and CD3OD
as solvent with TMS as an internal standard, δ in parts per million, and J in hertz. Mass spectra
were recorded using electron spray ionization (ESI-MS) or Fast atom bombardment spectra
(FAB-MS) using Argon/xenon as the FAB gas. HRMS spectra were recorded using ESI/Q-TOF
conditions. Optical rotation was determined by a Rudolph Research Analytical Autopol III
Automatic Polarimeter. Specific rotations [α]D25 for compound 26b is given in deg cm3 g−1 dm−1.
The purity of the compounds was determined by high performance liquid chromatography
(HPLC). Chiral HPLC of compound 26b was performed on W2998 instrument by using Chiral-
IA column; eluent A, propan-2-ol with 0.1% diethyl amine; eluent B, acetonitrile with 0.1%
19

diethyl amine, isocratic (10:90) with a flow rate of 0.6mL min−1 and detection at 280 nm; column
temperature of 25 °C; injection of 10 µL of 1 mM 26b.
General procedure for the synthesis of compound 25
To a stirred solution of intermediate aldehyde 24 (0.35 mmol) in dry THF (2 ml),
tetradecylmagnesium chloride 1M (0.43 mmol) was added dropwise under ice cooled condition.
After 5 min of stirring, ice was removed and the reaction mixture was stirred at rt for 4-5 h. After
completion of the reaction, it was quenched by slow addition of NH4Cl solution under ice cooled
condition. The aqueous layer was extracted with EtOAc (3x30 ml) and then the combined
organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated in vacuum.
Purification of the extracted mixture was done by silica gel (100-200 mess) column
chromatography (EtOAc/Hexane) afforded diastereomerically pure 25 with the syn configuration
as a solid.
tert-butyl ((2S,3S)-3-hydroxy-1-(4-methoxyphenyl)octan-2-yl)carbamate(25)
1
Solid; yield 73%; mp 73 °C; Rf (30% EtOAc/Hexane) 0.71; H NMR (400 MHz, CDCl3) 7.13
(2H, J= 8.1 Hz, d, Ar-H), 6.8 (2H, J= 8.4 Hz, d, Ar-H), 3.78 (3H, s, -OCH3), 3.69-3.61 (1H, m, -
CH), 3.54 (1H, m, -CH), 2.81-2.80 (2H, m, -CH2), 1.93 (1H, s, -OH), 1.40-1.23 (33H, m, tBu-H,
aliphatic H), 0.87 (3H, J= 6.3 Hz, t, -CH3); 13C NMR (100 MHz, CDCl3) 158.17, 156.15, 130.20,
113.91, 79.25, 71.67, 63.10, 55.24, 37.77, 34.60, 32.82, 31.92, 29.66, 29.57, 29.35, 28.35, 25.68,
22.68, 14.10, 1.01; m/z (ESI) 363.1 (100%), 364.1 (20%), 509.0 (3%, MCH3OH+); HRMS (ESI):
MH+ found 378.3382 C29H51NO4 requires 477.3818 (because of removal of Boc group).
General procedure for the synthesis of compound 26b
To the compound 25, 4N HCl in dioxane was added dropwise under ice cooled condition and
stirred for 0.5 hr at the same temperature. After completion of the reaction, solvent was
evaporated under reduced pressure. Then water was added to the residue and extracted with
EtOAc thrice (3x30 ml). The combined organic layer was basified with NaHCO3 two times,
washed with brine and dried over anhydrous Na2SO4 and concentrated under reduced pressure
and subjected to silica gel column chromatography to obtain the final compound 26b as
diastereomerically pure with the syn configuration in major amount (65 % yield) with some
demethylated product 26a (minor).To reduce the minor product 26a, Boc deprotection with
Dowex® 50Wx8 resin in MeOH was done and 26b as the major product (85 % yield) was
formed and only 5% of 26a was observed. The obtained compound 26b was optically pure with
specific rotation {[α]D25 -10.0 (c 0.05, MeOH).
(2S,3S)-2-amino-1-(4-methoxyphenyl)octan-3-ol (26b)
Solid; yield 85%; mp 64 °C; Rf (5% MeOH/CHCl3) 0.32; 1H NMR (400 MHz, CDCl3) 7.18 (2H,
J= 7.8Hz, d, Ar-H), 6.80 (2H, J= 7.8Hz, d, Ar-H), 6.16 (2H, br s, -NH2), 3.74 (3H, s, -OCH3),
3.60-3.26 (2H, m, -(CH)2), 2.94-2.87 (2H, m, -CH2), 1.25 (26H, m, aliphatic H), 0.87 (3H, J= 6.0
20

Hz, t, -CH3); 13C NMR (100 MHz, CDCl3) 158.66, 130.51, 128.10, 114.28, 70.87, 57.63, 55.15,
36.00, 34.05, 31.93, 29.75, 29.69, 29.58, 29.38, 25.53, 22.68, 14.10; m/z (ESI) 378.1 (100%,
MH+) 379.2 (20%); HRMS (ESI): MH+ found 378.3354 C24H43NO2 requires 378.3372. HPLC
analysis: retention time = 1.870 min; peak area, 98.63%; eluent A, propan-2-ol with 0.1% diethyl
amine; eluent B, acetonitrile with 0.1% diethyl amine, isocratic (10:90) over 30 min with a flow
rate of 0.6 mL min−1 and detection at 280 nm; column temperature of 25 °C. Specific rotation:
(2S, 3S)-26b{[α]D25 -10.0 deg cm3 g−1 dm−1 (c 0.05 g cm−3, MeOH)}
General procedure for the synthesis of compound 30
To an ice cooled solution of ester 29 (4.37 mmol) in dry THF (10 ml), LiAlH4 (5.24 mmol) was
added slowly, and allowed to stirred at rt for 1 h. After completion of the reaction, it was
quenched by slow addition of EtOAc and water, the aqueous layer was extracted with EtOAc (3x
30 ml), the combined organic layer was washed with brine, dried over anhydrous Na2SO4 and
solvent was removed under reduced pressure to furnish the alcohol compound 30. The crude
product was purified over 100-200 mess silica gel column chromatography by using
EtOAc/Hexane as an eluent.
(R)-tert-butyl (1-((tert-butyldiphenylsilyl)oxy-3-hydroxypropan-2-yl)carbamate (30)
1
Solid; yield 88 %; mp 74 °C; Rf (30% EtOAc/Hexane) 0.42; H NMR (400 MHz, CDCl3) 7.64
(4H, J= 7.5 Hz, d, Ar-H), 7.42-7.34 (6H, m, Ar-H), 3.79-3.67 (5H, m, -(CH2)2, -CH), 2.86 (1H, br
s, -OH), 1.42 (9H, s, tBu-H), 1.06 (9H, s, tBu-H); 13C NMR ( 100 MHz, CDCl3) 156.14, 135.57,
133.04, 132.99, 129.93, 127.88, 79.59, 63.87, 63.14, 53.25, 28.44, 26.93, 19.28; m/z (ESI) 429.6
(100%), 430.8 (22%, MH+); HRMS (ESI): MH+ found 430.2417 C24H37NO4Si requires
430.2414.
General procedure for the synthesis of compound 32
To the solution of previously prepared aldehyde 31 (0.23 mmol) in dry THF (2ml),
tetradecylmagnesium chloride 1M (0.35 mmol) was added slowly at 0 °C and was allowed to stir
at rt for 4-5 hr. After completion, the reaction was quenched by addition of NH4Cl solution and
water layer was extracted with EtOAc (3x30ml). The combined organic layer was washed with
brine solution, dried over anhydrous Na2SO4 and solvent was concentrated under reduced
pressure. The crude product was purified over silica gel column chromatography to furnish the
diastereomerically pure compound 32 with the syn configuration.
tert-butyl ((2S,3S)-1-((tert-butyldiphenylsilyl)oxy)-3-hydroxyoctan-2-yl)carbamate (32)
Oil; yield 81%; Rf(30% EtOAc/Hexane) 0.7; 1H NMR (400 MHz, CDCl3) 7.72-7.63 (4H, m, Ar-
H), 7.45-7.34 (6H, m, Ar-H), 3.93-3.81 (2H, m, -(CH)2), 3.62-3.59 (2H, m, -CH2), 2.95 (1H, br s,
t
-OH), 1.43 (9H, s, tBu-H), 1.25 (26H, m, aliphatic H), 1.06 (9H, s, Bu-H), 0.87 (3H, J= 6.6 Hz,
21

13
t, -CH3); C NMR (100 MHz, CDCl3) 156.09, 135.58, 135.54, 134.83, 132.72, 129.97, 129.51,
127.88, 127.64, 79.27, 72.67, 66.65, 63.00, 53.97, 33.85, 32.80, 31.94, 29.70, 29.46, 29.37,
28.40, 26.89, 26.77, 26.59, 25.77, 25.59, 22.70, 19.17, 14.12; m/z (ESI) 626.0 (100%), 627.0
(30%, MH+); HRMS (ESI): MH+ found 626.4608 C38H63NO4Si requires 626.4605.
General procedure for the synthesis of compound 33
To an ice cooled solution of synthesized compound 32 (1.59 mmol) in dry DCM (15 ml), DIPEA
(9.58 mmol) was added and stirred for 5 min at 0 °C. After 5 min, MOM-Cl (6.38 mmol) was
added slowly and then allowed to stir at rt for 3 h. After completion of the reaction, water was
added to the reaction mixture and aqueous layer was extracted with CH2Cl2 (3x30ml), the
combined organic layer was washed with brine solution, dried over anhydrous Na2SO4, and
solvent was removed under reduced pressure and the crude product was subjected to silica gel
column chromatography to furnish the compound 33.
tert-butyl ((5S,6S)-10,10-dimethyl-5-pentyl-9,9-diphenyl-2,4,8-trioxa-9-silaundecan-6-
yl)carbamate (33)
Oil; yield 79%; Rf (10% EtOAc/Hexane) 0.5; 1H NMR (400 MHz, CDCl3) 7.66-7.64 (4H, m, Ar-
H), 7.42-7.35 (6H, m, Ar-H), 4.56 (2H, s, -CH2), 3.84-3.82 (2H, m, -(CH)2), 3.68-3.57 (2H, m, -
t
t
CH2), 3.27 (3H, s, -CH3), 1.41 (9H, s, Bu-H), 1.25 (26H, m, aliphatic H), 1.05 (9H, s, Bu-H),
0.87 (3H, J= 6.5Hz, t, -CH3); 13C NMR (100 MHz, CDCl3) 155.67, 135.61, 133.38, 129.68,
127.70, 96.44, 78.96, 63.29, 55.70, 53.63, 31.93, 29.69, 29.36, 28.38, 26.84, 25.47, 22.69, 19.22,
14.11, 1.01; m/z (ESI) 669.9 (100%), 671.0 (35%, MH+); HRMS (ESI): MH+ found 670.4857
C40H67NO5Si requires 670.4867.
General procedure for the synthesis of compound 34
To a stirred solution of compound 33 (0.37 mmol) in dry THF (2 ml), 1M TBAF ( 0.55 mmol)
was added at 0 °C and allowed to stir at rt for 4-5 h. After completion of the reaction, water was
added to the reaction mixture and aqueous layer was extracted with CH2Cl2 (3x30ml), the
combined organic layer was washed with brine, dried over anhydrous Na2SO4, and solvent was
concentrated under reduced pressure to afford the compound 34. Purification was done by using
100-200 mesh silica gel column chromatography.
tert-butyl ((2S,3S)-1-hydroxy-3-(methoxymethoxy)octan-2-yl)carbamate(34)
1
Oil; yield 78%; Rf(30% EtOAc/Hexane) 0.4; H NMR (400 MHz, CD3OD) 4.68 (2H, s, -CH2),
t
3.76-3.66 (2H, m, -(CH)2), 3.65-3.51 (2H, m, -CH2), 3.39 (3H, s, -CH3), 1.46 (9H, s, Bu-H),
1.31 (26H, m, aliphatic H), 0.92 (3H, J= 6.4Hz, t, -CH3); 13C NMR (100 MHz, CD3OD) 156.84,
96.19, 78.77, 77.98, 76.47, 61.07, 60.75, 54.73, 54.12, 31.69, 31.01, 29.38, 29.30, 29.20, 29.09,
22

27.40, 25.12, 22.35, 13.08; m/z (ESI) 431.8 (100 %), 432.9 (20 %, MH+); HRMS (ESI): MH+
found 432.3700 C24H49NO5 requires 432.3689.
General procedure for the synthesis of compounds 36a-f
To an ice cooled solution of intermediate 35 (0.19 mmol) in acetonitrile (3 ml), RNH (0.58
mmol, various secondary amines) were added and subjected to reflux for 5-6 h. After completion
of the reaction, solvent was evaporated and water was added to the residue, the aqueous layer
was extracted with EtOAc (3x30 ml). The combined organic layer was washed with brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was
subjected to silica gel column chromatography to furnish the desired compounds 36a-f as
diastereomerically pure compounds with syn-stereochemistry in good yields 64-72%.
tert-butyl ((2S,3S)-1-(diethylamino)-3-(methoxymethoxy)octan-2-yl)carbamate (36a)
1
Solid; yield 72 %; mp 57 °C; Rf (30% EtOAc/Hexane) 0.15; H NMR (400 MHz, CDCl3) 6.04
(1H, s, -NH), 4.71-4.66 (3H, m, -CH2, -CH), 4.45-4.39 (2H, m, -CH2), 4.11-4.07 (1H, m, -CH),
3.89-3.83 (1H, m, -CH), 3.45-3.38 (6H, m, -CH3, -CH2, -CH), 1.25 (41 H, m, aliphatic H), 0.87
(3H, J= 6.5 Hz, t, -CH3); 13C NMR (100 MHz, CDCl3) 159.31, 97.29, 96.55, 81.81, 78.56, 66.67,
55.90, 55.74, 55.00, 31.91, 30.70, 29.64, 29.53, 29.48, 29.34, 24.94, 22.67, 14.09; m/z (ESI)
180.1 (100 %), 317.0 (5 %).
tert-butyl ((2S,3S)-1-(diisopropylamino)-3-(methoxymethoxy)octan-2-yl)carbamate (36b)
1
Solid; yield 69 %; mp 54 °C; Rf (30% EtOAc/Hexane) 0.16; H NMR (400 MHz, CDCl3) 6.36
(1H, s, -NH), 4.75-4.67 (2H, m, -CH2), 4.43-4.41 (2H, m, -(CH)2), 4.13-4.12 (1H, m, -CH2),
3.91-3.88 (1H, m, -CH2), 3.46-3.38 (5H, m, -CH2, -CH3), 1.42-1.25 (47H, m, tBu-H, aliphatic H),
0.90 (3H, J= 8Hz, t, -CH3); 13C NMR (100 MHz, CDCl3) 159.68, 97.19, 81.39, 78.54, 66.71,
55.88, 55.66, 31.90, 30.63, 29.66, 29.63, 29.60, 29.53, 29.48, 29.33, 24.97, 22.66, 14.08; m/z
(ESI) 437.2 (100 %), 515.4 (22 %. MH+); HRMS (ESI): MH+ found 515.4793 C30H62N2O4
requires 515.4788.
tert-butyl ((2S,3S)-1-(dibutylamino)-3-(methoxymethoxy)octan-2-yl)carbamate(36c)
1
Solid; yield 71 %; mp 50 °C; Rf (30% EtOAc/Hexane) 0.17; H NMR (400 MHz, CDCl3) 6.27
(1H, s, -NH), 4.69-4.66 (3H, m, -CH2), 4.43-4.39 (1H, m, -CH), 4.12-4.09 (1H, m, -CH), 3.89-
3.87 (2H, m, -CH2), 3.45-3.38 (6H, m, -CH3, -CH2, -CH), 1.41-1.25 (49H, m, aliphatic H), 0.87
(9H, J= 6.5 Hz, t, -(CH3)3); 13C NMR (100 MHz, CDCl3) 159.50, 97.23, 96.57, 81.56, 78.55,
66.69, 55.88, 55.68, 31.90, 30.65, 29.67, 29.66, 29.63, 29.60, 29.53, 29.48, 29.33, 24.97, 22.67,
14.09; m/z (ESI) 543.3 (100 %), 544.3 (30 %, MH+); HRMS (ESI): MH+ found 543.5114
C32H66N2O4 requires 543.5101.
tert-butyl ((2S,3S)-3-(methoxymethoxy)-1-thiomorpholinooctan-2-yl)carbamate(36d)
23

1
Solid; yield 67 %; mp 53 °C; Rf (30% EtOAc/Hexane) 0.14; H NMR (400 MHz, CDCl3) 6.25
(1H, s, -NH), 4.69-4.67 (3H, m, -CH2, -CH), 4.43-4.39 (2H, m, -CH2), 4.12-4.08 (1H, m, -CH),
3.90-3.85 (1H, m, -CH), 3.52-3.50 (1H, m, -CH), 3.45-3.38 (6H, m, -CH3, -CH2, -CH), 2.78-
2.72 (1H, m, -CH), 2.05-2.00 (1H, m, -CH), 1.41 (35H, m, aliphatic H), 0.87 (3H, J= 6.5 Hz, t, -
CH3); 13C NMR (100 MHz, CDCl3) 159.49, 97.23, 81.55, 66.68, 55.88, 55.68, 46.75, 31.90,
30.65, 29.63, 29.53, 29.48, 29.33, 24.96, 22.66, 14.09; m/z (ESI) 357.9 (100 %), 517.2 (75 %),
518.2 (23 %, MH+); HRMS (ESI): MH+ found 517.4045 C28H56N2O4Srequires 517.4039
tert-butyl ((2S,3S)-3-(methoxymethoxy)-1-(piperidin-1-yl)octan-2-yl)carbamate (36e)
1
Solid; yield 68 %; mp 50 °C; Rf (30% EtOAc/Hexane) 0.15; H NMR (400 MHz, CDCl3) 6.16
(1H, s, -NH), 4.69(3H, m, -CH2, --CH), 4.43-4.39 (1H, m, -CH), 4.11-4.08 (1H, m, -CH), 3.90-
3.84 (1H, m, -CH), 3.45-3.38 (6H, m, -CH3, -CH2, -CH), 3.19-3.17 (1H, m, -CH), 1.41-1.25 (41H,
t
m, Bu-H, aliphatic H ), 0.87 (3H, J= 6.5 Hz, m, -CH3); 13C NMR (100 MHz, CDCl3) 159.38,
97.27, 81.71, 66.67, 55.89, 55.72, 46.75, 31.91, 30.68, 29.67, 29.66, 29.64, 29.62, 29.60, 29.53,
29.48, 29.34, 25.36, 24.96, 22.67, 14.09; ; m/z (ESI) 288.26 (18 %), 316.29 (8 %, MH+).
tert-butyl((2S,3S)-3-(methoxymethoxy)-1-(4-methylpiperazin-1-yl)octan-2-yl)carbamate
(36f)
1
Solid; yield 64 %; mp 41°C; Rf (30% EtOAc/Hexane) 0.12; H NMR (400 MHz, CDCl3) 6.19
(1H, s, -NH), 4.70-4.66 (5H, m, -CH), 4.43-4.39 (2H, m, -CH2), 4.12-4.08 (2H, m, -CH2), 3.90-
3.85 (2H, m, -CH2), 3.45-3.38 (9H, m, -(CH3)2, -CH2, -CH), 1.29-1.25 (35H, m, aliphatic H),
13
0.88 (3H, J= 5.4 Hz, t, -CH3); C NMR (100 MHz, CDCl3) 159.38, 97.26, 81.68, 66.67, 55.89,
55.71, 31.91, 30.67, 29.64, 29.53, 29.48, 29.34, 24.96, 22.67, 14.09; m/z (ESI) 356.0 (100 %),
431.9 (15 %).
General procedure for the synthesis of compounds (37a-d)
To an ice cooled solution of 36a-d (0.21 mmol) in dry DCM (2 ml), BF3.OEt2 (0.25 mmol) was
added slowly and stirred for 5 min. After 5 min, ethane dithiol (0.25 mmol) was added and
stirred at rt for 2 h. After completion, water was added to the reaction mixture, the aqueous layer
was extracted with CH2Cl2 (3x30 ml). The combined organic layer was washed with brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure. Purification of the crude
product by silica gel column chromatographaffordeddiastereomerically pure compounds 37a-d
in good yields (69-72 %).
tert-butyl ((2S,3S)-1-(diethylamino)-3-hydroxyoctan-2-yl)carbamate(37a)
1
Solid; yield 71 %; mp 80 °C; Rf (50% EtOAc/Hexane) 0.2; H NMR (400 MHz, CDCl3) 6.37
(1H, s, -NH), 4.46-4.38 (2H, m, -CH2), 4.20-4.09 (2H, m, -CH2), 3.83-3.67 (2H, m, -CH2), 3.48
(1H, s, -CH), 3.05-2.96 (1H, m, -CH), 1.76 (1H, s, -OH), 1.50-1.25 (43 H, s, aliphatic H ), 0.87
24

(3H, J= 6.36 Hz, t, -CH3); 13C NMR (100 MHz, CDCl3) 160.60, 73.02, 67.36, 57.35, 33.25,
31.92, 29.69, 29.66, 29.61, 29.56, 29.50, 29.35, 25.71, 25.48, 22.68, 14.10; m/z (ESI) 258.2 (60
%), 288.3 (55 %), 385.0 (45 %), 414.4 (42 %), 474.2 (100 %, MCH3OH+).
tert-butyl ((2S,3S)-1-(diisopropylamino)-3-hydroxyoctan-2-yl)carbamate(37b)
1
Solid; yield 69 %; mp 83 °C; Rf (50% EtOAc/Hexane) 0.21; H NMR (400 MHz, CDCl3) 6.71
(1H, s, -NH), 4.45-4.41 (1H, m, -CH), 4.19-4.16 (1H, m, -CH), 3.83-3.68 (2H, m, -CH2), 3.41
(2H, s, -(CH)2), 2.13-2.07 (1H, m, -OH), 1.50-1.11(47H, m, aliphatic H), 0.87 (3H, J= 6.11 Hz, t,
-CH3); 13C NMR (100 MHz, CDCl3) 160.60, 73.02, 67.36, 57.35, 33.25, 31.92, 29.69, 29.66,
29.61, 29.56, 29.50, 29.35, 25.71, 25.48, 22.68, 14.10; m/z (ESI) 300.6 (58 %), 318.0 (100 %),
371.4 (55 %), 502.2 (45 %, MCH3OH+).
tert-butyl ((2S,3S)-1-(dibutylamino)-3-hydroxyoctan-2-yl)carbamate(37c)
1
Solid; yield 71 %; mp 81°C; Rf (50% EtOAc/Hexane) 0.22; H NMR (400 MHz, CDCl3+
CD3OD) 4.46-4.39 (2H, m, -CH2), 4.19-4.17 (2H, m, -CH2), 3.88-3.76 (2H, m, -CH2), 3.56-3.45
t
(2H, m, -(CH)2), 1.53-1.35 (46H, m, Bu-H, aliphatic H), 0.88 (6H, m, -CH3); 13C NMR ( 100
MHz, CDCl3+ CD3OD) 164.63, 76.57, 71.12, 61.23, 36.78, 35.77, 33.52, 33.49, 33.45, 33.41,
33.39, 33.19, 31.15, 29.34, 26.51, 17.85; m/z (ESI) 358.3 (15 %), 386.5 (28 %), 398.5 (30 %),
414.5 (15 %), 485.0 (40 %), 530.2 (40 %, MCH3OH+).
tert-butyl ((2S,3S)-3-hydroxy-1-thiomorpholinooctan-2-yl)carbamate(37d)
1
Solid; yield 72 %; mp 94 °C; Rf (50% EtOAc/Hexane) 0.21; H NMR (400 MHz, CDCl3) 6.56
(1H, s, -NH), 4.45-4.38 (2H, m, -CH2), 4.20-4.17 (2H, m, -(CH)2), 3.84-3.68 (2H, m, -CH2), 3.48
(2H, s, -CH2), 3.29-3.03 (2H, m, -CH2), 2.17-188 (2H, m, -CH2), 1.50-1.48 (1H, m, -OH), 1.27
(35H, m, aliphatic H) 0.88 (3H, J= 6.2 Hz, t, -CH3); 13C NMR (100 MHz, CDCl3) 160.47, 73.03,
67.32, 57.29, 33.28, 31.92, 29.69, 29.65, 29.60, 29.54, 29.49, 29.35, 25.67, 25.46, 22.68, 14.10;
m/z (ESI) 386.1 (30 %), 394.2 (100 %), 398.5 (30 %).
Biological Assays:
Cell Culture
Various human cancer cell line including DLD-1, HT-29 (colorectal adenocarcinoma) A-549,
NCI-H358 (lung carcinoma) MCF-7, MDA-MB-231(Breast cancer cell line), were obtained from
ATCC, USA. We used early passage cells and cultured according to manufacturer’s instructions.
Cells were plated at 40% confluency, after overnight incubation different drug concentrations
used for cell treatment as indicated in results section.
Evaluation of in vitro anticancer activity of spisulosine derivative
25

In-vitro cytotoxic efficacy of compounds against various cancer cell lines were assayed using
standard sulforhodamine B (SRB) cytotoxicity assay53,54. Absorbance was measured on a plate
reader (epoch microplate reader, biotek) at 510 nm to calculate the percent inhibition in cell
growth by using the formula: [100(absorbance of compound treated cells/absorbance of
untreated cells)] 100.
Visualization of MDC vacuoles
DLD-1 colon cancer cells were incubated in the presence of drug for 20 hours and labeled with
50mM concentration of autofluorescent marker monodensylcadaverine MDC (Sigma Chemical55
Co.) in PBS at 37°c for 20 min in dark, after washing with PBS cells were immediately analyzed
by a laser scanning confocal microscopy, (excitation 390nm; emission 460nm)
Western blot analysis
After treatment cells were lysed in NP-40 buffer supplemented with phosphatase and protease
inhibitors cocktail. Lysates were quantitated using pierce BCA protein assay kit. Equal amount
of protein in SDS-PAGE sample buffer were loaded in 15% SDS polyacrylamide gel and
tranfered to PVDF membranes. Immunoreactivity was detected by enhanced chemiluminescence
solution (immobilon western ECL, Millipore) after incubation of membrane with recommended
amount of primary and secondary. Beclin-1 (#3495), LC3A/B (#12741), Atg5 (#12994), Atg3
(#3415), Atg7 (#8558), cleaved PARP (#5625) were purchased from cell signaling technology
and GAPDH (#25778) antibody were purchased from Santa Cruz biotechnology.
Apoptosis analysis by Flowcytometry
Different stages of apoptosis were distinguished using an Annexin-V-FITC/Propidium Iodide
apoptosis kit (BD Biosciences). After overnight incubation DLD1- cells were treated for 24h in
presence of compound 26b and spisulosine, For Annexin-V staining cells were washed by PBS
and resuspended in 1x binding buffer provided by Sigma then treated and vehicle control cells
were stained with FITC conjugated Annexin-V and propidium iodide and incubated for 20 min
in dark followed by washing with 1x binding buffer. The stained cells were acquired in a FACS
caliber (Becton Dickinson) and analyzed by FlowJo software.
Confocal Microscopy
DLD-1 colon cancer cells were grown on coverslips for overnight and the next day after
treatment cells were fixed with 4% paraformaldehyde in PBS and permeabilized by 0.1%
26

TritonX-100 for 10 minutes followed by blocking with 2% BSA for 45 minutes at RT. After
overnight primary antibody incubation at 4°C washed cells were then incubated with fluorescent-
conjugated secondary antibodies at RT for 2 hours, followed by DAPI staining for 5 min at RT.
After washing, cells were mounted with anti-fade mounting medium on glass slides and viewed
under an inverted confocal laser scanning microscope (Zeiss Meta 510LSM; Carl Zeiss, Jena,
Germany). Plan Apochromat 63X/1.4 NA Oil DIC objective lens was used for imaging and data
collection. Appropriate excitation lines, excitation and emission filters were used for imaging.
Transmission Electron Microscopy (TEM)
Cells were fixed in 2.5% Glutaraldehyde in phosphate buffer, pH 7.4. Post-fixation was
performed in 1% OsO4 and encapsulated in agarose. Cells were subsequently dehydrated in
acending series of ethanol and embedded and polymerised in Spurr resin. Ultrathin sections were
obtained using a Leica UC7 ultramicrotome and double stained with uranyl acetate and lead
citrate. 150 mesh copper grids were used to pick up the sections. Grids were observed under a
Jeol JEM-1400 Transmission Electron Microscope at 80 kV after proper alignment. Images were
recorded using a Gatan Orius SC200 CC camera. At least 200 cells were analyzed from 4 grids
for each set.
5. SUPPORTING INFORMATION
The characterization data of all new compounds are attached.
6. ACKNOWLEDGMENTS
We thank Mr. Shashank Shekhar Mishra, and Mr. Ashok Kumar Sharma some initial preliminary
experiment of this manuscript respectively. Fellowship support to Asha Ganesher from CSIR and
Priyank Chaturvedi from DBT are acknowledged. Partial financial support from DST
(EMR/2016/006935), DBT (BT/AIR0568/PACE-15/18) and ICMR (2019-1350) to Dr. Datta as
well as to BRNS-BARC (3511410812018-BRNS/35032), Mumbai to Dr. Panda is highly
appreciated. Instrumental support from SAIF, CDRI is highly acknowledged. CDRI
communication no for this article is 93/2019/GP.
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