Efficient syntheses and anti-cancer activity of xenortides A-D including: Ent / epi -stereoisomers

Article abstract of DOI:10.1039/c8ob00452h

A one-pot, two-step, total synthesis of naturally occurring xenortides A, B, C and D, (Xens A-D) isolated from the bacterium Xenorhabdus nematophila, and an entire complementary set of stereoisomers, has been achieved. Compounds were synthesized utilizing an isocyanide-based Ugi 4-CR followed by facile N-Boc deprotection. The reaction sequence took advantage of the chiral pool of N-Boc protected amino acids (l-Leu/Val and d-Leu/Val) with aryl isocyanides, phenyl acetaldehyde and methylamine giving the desired Xens A-D (A and B >98% ee) and all subsequent stereoisomers in reasonable yields upon deprotection followed by separation of diastereomers. Also, detailed mechanistic insights for diastereoselectivity of (-)-Xen A, as a model in the Ugi 4-CR, has been described. Moreover, for the first time, this focused library was screened for cytotoxicity against a panel of epithelial cancer cell lines as well as normal cell lines with an MTT proliferation assay. The structure-activity relationship (SAR) study demonstrated that tryptamides Xen B and D were more active than phenylethylamides Xen A and C. Furthermore, (-)-Xen B (IC₅₀ = 19-25 μM) and ent-(+)-Xen D (IC₅₀ = 21-26 μM) gave the highest cytotoxicity and they were also found to be non-toxic toward normal cells. Importantly, the SAR results indicate that the stereochemistry at C₈ and C₁₁ in (-)-Xen B and ent-(+)-Xen D play a critical role in cytotoxic activity.

Full text of DOI:10.1039/c8ob00452h

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Maddirala, N. A. Hussein, H. A. Amawi, A. Tiwari and P. R. Andreana, Org. Biomol. Chem., 2018, DOI:
10.1039/C8OB00452H.
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Efficient Syntheses and Anti-Cancer Activity of Xenortides A-D
Including ent/epi-Stereoisomers
N. Esmatia, A. R. Maddiralaa, N. Husseinb, H. Amawib,#, A. K. Tiwarib and P. R. Andreana*a
A one-pot, two-step, total synthesis of naturally occurring Xenortides A, B, C and D, (Xens A-D) isolated from the bacterium
Xenorhabdus nematophila, and an entire complementary set of stereoisomers, has been achieved. Compounds were
synthesized utilizing an isocyanide-based Ugi 4-CR followed by facile N-Boc deprotection. The reaction sequence took
advantage of the chiral pool of N-Boc protected amino acids (L-Leu/Val and D-Leu/Val) with aryl isocyanides, phenyl
acetaldehyde and methylamine giving the desired Xens A-D (A and B >98% ee) and all subsequent stereoisomers in
reasonable yields upon deprotection followed by separation of diastereomers. Also, detailed mechanistic insights for
diastereoselectivity of (-)-Xen A, as a model in the Ugi 4-CR, has been described. Moreover, for the first time, this focused
library was screened for cytotoxicity against a panel of epithelial cancer cell lines as well as normal cell lines with an MTT
proliferation assay. The structure–activity relationship (SAR) study demonstrated that tryptamides Xen B and D were more
active than phenylethylamides Xen A and C. Furthermore, (-)-Xen B (IC50 = 19-25 µM) and ent-(+)-Xen D (IC50 = 21-26 µM)
gave the highest cytotoxicity and they were also found to be non-toxic toward normal cells. Importantly, the SAR results
indicate that the stereochemistry at C8 and C11 in (-)-Xen B and ent-(+)-Xen D play a critical role in cytotoxic activity.
a.Department of Chemistry and Biochemistry and School of Green Chemistry and Engineering, The University of Toledo, 2801 W. Bancroft St, Toledo, OH 43606
b.Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, OH 43606, US
#. Current address: Department of Pharmacy Practice, College of Pharmacy, Yarmouk University, Irbid, Jordan 21163
Introduction
Cancer is the second leading cause of death globally and as
reported by the World Health Organization (WHO), cancer was
responsible for 8.8 million deaths in 2015.1 Despite an overall
positive progression in cancer chemotherapy, limited
improvements in patient survival has been documented in
many countries and death rates are increasing. Multidrug
resistance, lack of selective chemotherapeutic drugs against
target tumor cells and severe side effects are major limitations
for the availability of anticancer agents.2 The treatment of
cancer has been a serious concern in public health and urgent
development of novel classes of therapeutic agents or
anticancer drugs with improved efficacy are in high demand.3, 4
Peptides, compared to other small organic molecules and
proteinaceous immunotherapeutics, have several desirable
properties such as high activity, low immunogenicity, good
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biocompatibility, and they are readily amenable to synthetic stereochemical combination, which is standard for SAR
6
modifications.5, As such, peptides have become promising studies, following the mantra of medicinal and biological
molecules for anticancer agents.7-9 It is therefore important to chemistry. Our retrosynthetic analysis for those compounds is
strategize an efficient synthetic approach that can eliminate shown in Scheme 1
any unwanted material during the process to find an optimal
.
molecule consisting of the most appropriate structural
conformation.10
Results and Discussion
Chemistry
It is well-known that secondary bacteria and fungi metabolites
12
are valuable sources of chemotherapeutic agents.11,
Synthetic efforts commenced with synthesizing phenylethyl
isocyanide (1a24) and indole ethyl isocyanide (1b25) as required
Bacterial secondary metabolites, Xenortides (Xens) A-D
(Scheme 1), are a family of natural products extracted from
precursors in the Ugi 4-CR.
These compounds were
the bacterium Xenorhabdus nematophilus.13-15 Xen A and C
consist of N-phenylethylamide derivatives of the dipeptide (N-
Me-L-Leu and N-Me-L-Phe) and (N-Me-L-Val and N-Me-L-Phe),
respectively. The counterparts, (-)-Xen B and D, consist of
tryptamide derivatives of the dipeptide (N-Me-L-Leu and N-
Me-L-Phe) and (N-Me-L-Val and N-Me-L-Phe) respectively.
synthesized through common formylation26 and dehydration27,
28 procedures. Formylation was achieved via refluxing the aryl
ethylamine (1a′ and 1b′) with ethyl formate for 6 h. A solution
of the related aryl ethyl formate (1a
anhydrous dichloromethane was treated with POCl3 at -78 C
for to furnish the required isocyanides in nearly
quantitative yields Scheme 2
″ and 1b″) and Et3N in
o
3
h
Recent observations by Reimer et al. suggest that these
14
.
compounds have some anti-parasitic activities
In view of
aforementioned biological activities of naturally occurring Xens
A-D and our longstanding interest in eradicating cancer,16-19
exploring the potential anticancer activity of these and
derivative compounds, encouraged us to devise a facile
synthetic approach for naturally occurring Xen compounds and
a cohort of stereoisomers. Here within, we document our
synthetic strategy to Xens A-D including all possible
stereoisomers. Furthermore, we demonstrate compound bio-
potential by examining anticancer activity of each isomer in
several epithelial cancer cells. Moreover, this study represents
the first anticancer activity report on the Xen family.
Recently, the synthesis of (-)-Xen D was reported by Reimer
et.al.14 (-)-Xen D was synthesized via standard peptide
coupling reaction conditions using several expensive peptide
coupling reagents and multi-step syntheses giving rise to
several purification procedures.
Furthermore, the
aforementioned strategy would not be easily accessible to a
small library of derivatives as further synthetic manipulations
would be required. These limitations encouraged us to devise
a streamlined chemical route toward the synthesis of highly
enantiopure Xens. To the best of our knowledge, there are no
literature reports describing the synthesis of the complete set
of Xen natural products and their ent/epi-stereoisomers.
Moreover, we believe that the synthesis of highly enantiopure
natural Xens A-D and their related stereoisomers may shed
light on how the stereochemical properties of chiral, non-
racemic Xens play out in anticancer studies, which have never
been previously attempted. In the context of developing
simple structural manipulations of the starting materials and
atom-economical synthetic routes for the synthesis of Xens
and all their stereoisomers, we designed an efficient and
effective synthetic strategy employing an Ugi four component
reaction (4-CR) process.20-23 Our synthetic strategy, involving
only two sequential steps, accomplished in a single pot, allows
us to obtain the desired compounds in a highly stereoselective
fashion using readily available commercial starting materials.
As such, this approach is highly efficient, allowing for
Xenortide library development with every possible
Scheme 1. Retrosynthetic approach to naturally occurring Xens A-D using an Ugi
4-CR ( ) + )).
1(
a
,
b
2 + 3 + 4(a,b
Scheme 2. Formylation and dehydration for the synthesis of aryl isocyanides 1a
and 1b
.
Having precursors 1a and 1b in hand, we began our two-step
synthetic protocol of (-)-Xen A ( ) and epi-(+)-Xen A (5a
Scheme ). The reaction was designed to employ
stoichiometric equivalents of phenylethyl isocyanide (1a),
phenyl acetaldehyde ( ), methylamine ( ) (33% weight in
ethanol) and Boc-N-methyl-L-leucine 4a which readily
5
)
(
3
2
3
(
)
dissolved in methanol as the solvent. The reaction was then
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stirred at room temperature for 24 h. The resultant mixture
led to the formation of two diastereomers, which were readily
visualized using TLC; Rf values of 0.5 and 0.8. However, due to
the formation of rotamers during 1H NMR analysis, we were
unable to quantitate the actual diastereomeric ratios of the
unpurified mixture of 5′ and 5a′
. We have performed NMR
studies at high temperature in an attempt to clarify the
rotameric issue, however, complete coalescence was not
achieved. After careful separation of the Boc protected
diastereomers using silica gel column chromatography with
1
hexane and ethyl acetate as the eluents, H NMR analysis was
conducted for individual diastereomers 5′ and 5a′
.
Unfortunately, rotamers were once again problematic and
definitive NMR characterization continued to be elusive.
Compounds 5′ and 5a′ were then subjected to Boc
deprotection using 50% TFA in DCM at 0 °C for 2 h (Scheme 4).
After TLC indicated complete Boc deprotection, isolation and
purification of the individual (-)-Xen A (
5a) ensued using heptane and ethyl acetate as the solvents
with normal phase silica gel chromatography. Pure
5) and epi-(+)-Xen A
(
1
compounds were then subjected to H NMR spectral analysis
and at this point we did not observe rotamers, which had
plagued our ability to properly characterize other
intermediates as previously stated. All of the NMR spectral
analysis and the optical rotary values observed for compounds
Scheme 4. One-pot, two-step synthesis of
5 and 5a.
To further support a more efficient and effective process to
Xens A-D, the strategy was intended to avoid tedious workup
and purifications of Boc-protected compounds such as 5′ and
5a′.
5
and 5a gave identical peak matching to previous literature
reports.13, 14
In formalizing the one-pot, two-step synthetic process for (-)-
Xen A (5) and epi-(+)-Xen A (5a) (Scheme 4), we decided to
follow the exact route for the synthesis of the other Xen family
of natural products. Therefore, in all cases, when TLC and
mass spectral analysis indicated completion, methanol was
evaporated under reduced pressure and without any further
purification, the crude mixture was subjected to the Boc
deprotection conditions which consisted of 50% TFA in DCM at
0 °C followed by stirring for 2 h.
The unpurified mixture of (+/-)-Xens A-D was then analyzed
using 1H NMR to determine diastereomeric ratios and then
silica gel column chromatography with heptane and ethyl
acetate as the solvent system was used to isolate our target
molecules. Further NMR spectral analysis of the individually
isolated pure compounds was conducted and peak shift values
were observed matching exactly to those found in the
literature.13, 14
At this point in the study, we elected to conduct a detailed
investigation on the effect of temperature on
diastereoselectivity. Reactions were subjected to conditions at
0 °C, room temperature and 40 °C all the while maintaining
consistency with a 24 h reaction time-frame using methanol as
the solvent. From these studies, we observed that room
temperature conditions gave the best diastereomeric ratios of
Scheme 3. Two-step synthesis of (-)-Xen A (5) and epi-(+)-Xen A (5a).
To by-pass the rotameric issue that we encountered with the
initial two-step sequence and our problem quantifying
1
diastereomeric ratios of intermediates using H NMR analysis,
1.5:1 when compounds 1a
All the other natural Xens and their stereoisomers were
obtained employing the conditions noted in entry 2; Table 1
, 2, 3, 4a were used (Table 1).
we elected to alter our synthetic strategy and re-design our
approach that would include a one pot, two-step synthetic
process (Scheme 4).
.
Using the noted conditions enabled us to access a small library
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of Xen derivatives with isocyanides 1a and 1b along with L-Leu 1H NMR and Rf values of each diastereomer in heptane/ethyl
and L-Val. Diastereomeric ratios were again determined using acetate (4:1) were noted as documented in Table 2
.
Table 1. Effect of temperature on the one-pot synthesis.
diastereomeric ratio
a[5 (S,S):5a (S,R)]
1.3:1
byield (%)
5 (S,S)
15
byield (%)
5a (S,R)
<5
entry
conditions
1
2
3
MeOH, 0 °C, 24 h
MeOH, r.t., 24 h
MeOH, 40 °C, 24 h
1.5:1
27
18
1.1:1
17
13
aDiastereomeric ratios determined using 1H NMR analysis of the unpurified reaction mixture (T1 = 3.5). bCompounds purified using silica gel
chromatography with heptane and ethyl acetate as the solvents.
Table 2. A one-pot, two-step reaction sequence for (+/-) and epi-(+/-)-Xens.
entry
R
R′
aproduct
)/epi-(+)-Xen A (5a
bdiastereomeric ratio
Rf (cm)
1
Ph
(L-Leu) - 4a
(-)-Xen A (
5
)
1.5:1.0
0.5/0.8
2
3
4
5
6
7
8
3-Indole
Ph
(L-Leu) - 4a
(L-Val) - 4b
(L-Val) - 4b
(D-Leu) - 4c
(D-Leu) - 4c
(D-Val) - 4d
(D-Val) - 4d
(-)-Xen B (
(-)-Xen C (
(-)-Xen D (
6
7
8
)/epi-(+)-Xen B (6a
)/epi-(+)-Xen C (7a
)
)
1.5:1.0
1.3:1.0
1.3:1.0
1.0:1.3
1.0:1.4
1.0:1.1
1.0:1.5
0.3/0.6
0.5/0.7
0.4/0.7
0.3/0.6
0.5/0.8
0.5/0.8
0.4/0.6
3-Indole
Ph
)/epi-(+)-Xen D (8a)
ent-(+)-Xen A (5b)/ent-epi-(-)-Xen A (5c
ent-(+)-Xen B (6b)/ent-epi-(-)-Xen B (6c
)
3-Indole
Ph
)
ent-(+)-Xen C (7b)/ent-epi-(-)-Xen C (7c
)
3-Indole
ent-(+)-Xen D (8b)/ent-epi-(-)-Xen D (8c
)
aCompounds purified using silica gel chromatography with heptane and ethyl acetate as the solvents. bDiastereomeric ratios determined using 1H NMR analysis of the
crude
reaction
mixture.
In all cases, formation of diastereomers with (S) configuration
at C8 were favored over those with the (R) configuration
(
Scheme 5).29-32 We assume that preferable formation of the
S-diastereomer in all synthesized compounds can be explained
in the detailed mechanism of (-)-Xen A ( ) as a model in the
5
Ugi 4-CR. In this mechanism, we propose that nucleophilic
attack by the phenylethyl isocyanide (1a) occurs from the Re-
face of the thermodynamically more stable (E)-imine, which
forms first in the equilibrium sequence of events between
and ). We argue that this is made possible through
hydrogen-bond donation of amino acids 4a 4d, which block
2
3 (I
-
the Si-face of the (E)-imine and leads to the slightly preferred
(S) configuration at C8 (II). What follows next is attack on the
nitrilium ion by the carboxylate leading to intermediate (III
)
which undergoes the Mumm rearrangement30 in the rate
determining step giving 5′, which, when treated with TFA in
DCM renders (-)-Xen A (
purification.
5) as the major diastereomer after
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H
CH3
H
I
O
Boc
N
N
H
O
N
Ph
CH3
H
Cytotoxicity Evaluation and the Structure Activity Relationships
Study
N
H
HO
CH3
O
NH2
R'
R'
4a-4d
CH3
3
O
2
+
+
C
N
1a
R"
Boc
Ph
The influence of chirality at C8 and C11 on cytotoxicity of
naturally occurring Xens A-D and all their stereoisomers
(enantiomers and diastereomers) were investigated and some
interesting results were observed from this focused library of
compounds. Tryptamides Xen B and D stereoisomers were
overall more active than the phenylethylamides Xen A and C
(E)-imine
CH3 Ph
Re-face attack
Mumm Rearrangement
Ph
R" =
H3C
N
H
III
N
R"
O
II
CH3
O
C8
H
H
H
N
Boc
N
H
O
Ph
O
CH3
C8
(S)
C11
stereoisomers.
It was also previously reported that
R'
CH3
H
C
N
R"
R'
N
tryptamides (-)-Xen B and D, natural products were more
active than natural phenylethylamides (-)-Xen A and C against
sleeping sickness (Trypanosoma brucei rhodesiense) and
malaria (Plasmodium falciparum) diseases.14 These data
together suggest that tryptamide analogues play a significant
role in the enhancement of biological activity of the Xen
family.
Ph
Boc
O
Boc
O
H
TFA/DCM
0 °C, 2h
H3C
N
C11
H3C
H
N
N
N
CH3
N
CH3
C11
H
C8
Ph
C8
N
Ph
Ph
O
Ph
O
5
When R' = L-Leu - 5'
In addition, (-)-Xen B and ent-(+)-Xen D were found to be most
active among all tested compounds. (-)-Xen B at 19-25 µM
produced significant morphological changes against a total of
five tested cancer cell lines, HCT-116, H460, OV-2008, MDA-
MB-23, DU-145 (Figure 1). Also, ent-(+)-Xen D had a 21-26 µM
cytotoxicity, selective against three tested cancer cell lines that
Scheme 5. Plausible mechanism for diastereoselectivity in the Ugi 4-CR.
Under the conditions noted, all desired target products were
obtained in fair to moderate yields. We were able to calculate
the enantioselectivities for (-)-Xen A/B and ent-(+)-Xen A/B
using specific optical rotation values of those compounds
reported in literature2 (Table 3). The excellent optical rotary
values observed between our compounds and those published
reveal that retention of chirality is maintained in the N-Boc
protected amino acids (L/D-Leu) under our protocol. This
further validates that under our noted reaction conditions no
racemization at C11 occurred.
included HCT-116, OV-2008 and MDA-MB-23.
Moreover,
inspecting the cytotoxicity of active compounds ((-)-Xen B and
ent-(+)-Xen D), in comparison to their related stereoisomers,
revealed that although they have the same chemical
structures, they exhibit distinct differences in anticancer
activities. For example, natural (-)-Xen B in comparison to its
enantiomer, ent-(+)-Xen B, revealed markedly more cytotoxic
activity (IC50 = 19-25 µM) than its enantiomer (IC50 = 41-100
µM) in all evaluated cancer lines. Similarly, ent-(+)-Xen D
exhibited significantly higher cytotoxic activity than its
enantiomer (-)-Xen D, with IC50 values of 21-50 µM and > 100
µM, respectively. Furthermore, in this study, the potency of
the most active compounds was considerably greater for
cancer cells when compared to the normal cells, meaning that
no toxicity was recorded toward normal cell lines CRL-1459
and CHO. (These compounds were noted to be considerably
safe when tested on the normal cell lines.) (Figure 2).
Cisplatin, a well-known anticancer agent, was used as a
positive control in our experiments. We observed that
although, cisplatin killed the cancer cells at a concentration
range of 8-14 µM, they were less selective compared to Xen on
normal cells (Table 4).
Biological Studies
After completing the synthesis of Xen compounds noted in
Table 3, we turned our attention to cytotoxicity analyses using
five different cancer cell lines; colon carcinoma (HCT-116),
human lung carcinoma (H460), human ovarian carcinoma (OV-
2008), human breast adenocarcinoma (MDA-MB-231), and
human prostate adenocarcinoma (DU-145), as well as normal
cell lines human epithelial colon (CRL-1459) and Chinese
hamster ovarian cells (CHO) using the MTT assay.33,
34
The
antiproliferative efficacy data are presented as IC50 values
defined as the concentration of the compound that decreases
cell proliferation at 50% (Table 4). The IC50 values were
generated from at least three independent experiments. Cell
survival was determined by MTT assay as described in
materials and methods.
Table
3.
Library
of
natural
and
unnatural
Xens.
compound
name
ayield (%)
b % ee
5
(-)-Xen A
27
18
15
>98
5a
5b
epi-(+)-Xen A
ent-(+)-Xen A
̶
>98
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5c
6
ent-epi-(-)-Xen A
(-)-Xen B
19
28
19
20
28
25
19
21
23
20
15
17
25
̶
>98
6a
6b
6c
7
epi-(+)-Xen B
ent-(+)-Xen B
ent-epi-(-)-Xen B
(-)-Xen C
̶
>98
̶
̶
7a
7b
7c
8
epi-(+)-Xen C
ent-(+)-Xen C
ent-epi-(-)-Xen C
(-)-Xen D
̶
̶
̶
̶
8a
8b
8c
epi-(+)-Xen D
ent-(+)-Xen D
ent-epi-(-)-Xen D
̶
̶
̶
aAfter silica gel column chromatography. bEnantiomeric excess (ee) was determined using specific optical rotation values reported for (-)-Xen A and B.13
Figure 1. Morphological analysis of the
cytotoxic effects of (-)-Xen B at 0 and 19-25
µM on cancerous cell lines including human
colon carcinoma (HCT-116), human lung
carcinoma (H460), human ovarian carcinoma
(OV-2008), human breast adenocarcinoma
(MDA-MB-231),
human
prostate
adenocarcinoma (DU-145) and normal human
epithelial colon cell (CRL-1459). The cells
were photographed at 20 ˚C, 72
h after
incubation for each triplicate treatment.
Table 4. Cytotoxic effects of naturally occurring Xens A-D and stereoisomers on the survival of cancer cell lines and normal cell lines.
Colon
aHCT-116
Lung
aH460
Ovarian
aOV-2008
Breast
aMDA-MB-231
Prostate
aDU-145
Normal Cells
Compounds
aCRL
aCHO
(-)-Xen A
>100
>100
>100
95
>100
99
88
epi-(+)-Xen A
ent-(+)-Xen A
ent-epi-(-)-Xen A
(-)-Xen B
81
86
46
19
48
48
25
63
62
90
95
97
72
65
>100
88
94
98
67
25
67
67
42
72
85
99
>100
>100
>100
86
>100
78
78
58
60
87
24
25
21
54
epi-(+)-Xen B
ent-(+)-Xen B
ent-epi-(-)-Xen B
(-)-Xen C
67
59
55
63
78
>100
58
>100
60
41
>100
87
87
20
59
79
50
36
>100
>100
>100
92
epi-(+)-Xen C
ent-(+)-Xen C
75
72
59
92
97
97
97
>100
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ent-epi-(-)-Xen C
(-)-Xen D
>100
99
>100
>100
79
>100
>100
64
>100
>100
49
97
>100
85
>100
>100
>100
88
>100
76
epi-(+)-Xen D
ent-(+)-Xen D
ent-epi-(-)-Xen D
bCisplatin
54
26
35
25
21
50
>100
72
84
35
81
64
64
90
88
11
8
14
12
14
21
18
aThe IC50 (µM) values are represented as mean ± SD of three independent experiments performed in triplicate. bCisplatin was used as a positive control.
The Cytotoxicity Effects of Active (-)-Xen B and ent-(+)-Xen D
Moreover, ent-(+)-Xen D (highlighted in yellow) was selectively
active against three cancer cell lines, including HCT-116, MDA-
MB-231 and OV-2008. In those three cancer cell lines, ent-(+)-
on Various Cell Lines (Cancerous and Non-Cancerous Cells)
Based on our data, for (-)-Xen
B and the cohort of
Xen D with (R) configuration at C11 revealed better cytotoxic
stereoisomers, the chiral properties of C8 containing the (
S
)
effects than its diastereomer, epi-(+)-Xen D, with respective
stereo-configuration is considered a critical factor influencing
IC50 values of 21 – 35 µM and 49 – >79 µM. Similar results
compound potency. As summarized in Table 4, (-)-Xen B
were observed for ent-epi-(-)-Xen D and (-)-Xen D, proving that
(highlighted in yellow) with (S) configuration at C8, was
the stereochemistry of C11 in (-)-Xen D plays an important role
in cytotoxic activity.
determined to have excellent cytotoxic activities (IC50 = 19 – 25
µM) against five cancer cell lines, and was determined to be
approximately 2-fold more potent than its corresponding
diastereomer, epi-(+)-Xen B with the (
R) configuration at C8.
Coincident results were observed for ent
-epi-(-)-Xen B and ent
-
(+)-Xen B, with IC50 values of 20 – 60 µM and 41 – >100 µM,
respectively. These results strongly suggest that the chirality
at C8 plays a substantial role in cytotoxic activity of (-)-Xen B
stereoisomers.
(A)
(B)
Figure 2. (A) IC50 values and (B) cell survival curves of cancer cells compared to that of normal human epithelial colon cell (CRL-1459) when treated with (-)-Xen B
determined by the MTT assay.
IC50 values are represented as means ± SD of three independent experiments performed in triplicate.
enantiomeric excesses (>90% ee) in reasonable yields. Based
on our knowledge of the literature, the anticancer activities of
sixteen Xen family of natural products and all their subsequent
stereoisomers are reported here for the very first time. When
dealing with biological interactions especially for medicinal
applications, comparing the activity of stereoisomers is crucial,
Conclusion
In conclusion, a concise synthesis of natural Xen A-D and all
subsequent stereoisomers has been achieved in a one-pot,
two-step Ugi 4-CR synthetic strategy. In this reaction, chiral N-
Boc protected amino acids (L-Leu/Val and D-Leu/Val), aryl
isocyanides, phenyl acetaldehyde and methylamine were
utilized as starting materials. All reactions were carried out
efficiently and simple chromatographic purification led to
as biological targets are chiral.
The stereo-SAR study
demonstrated that only two compounds, among sixteen
synthesized, were very active in the MTT assay. Natural (-)-
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Xen B revealed high cytotoxic activity against five tested bath for 2 h. The reaction was monitored for completion by
cancer cell lines and ent-(+)-Xen indicated decent TLC and the contents were then concentrated under reduced
D
cytotoxicity, selectively against three cancer cell lines, pressure. The crude compound was then subjected to flash
described in Table 4. Furthermore, SAR results indicated that column chromatography (EtOAc/Hexanes) to yield pure
the stereochemistry at C8 and C11 in (-)-Xen B and ent-(+)-Xen compounds 5-8 (a-c).
D, respectively, plays an important role in cytotoxic activities.
(S)-N,4-dimethyl-2-(methylamino)-N-((S)-1-oxo-1-
Moreover, it should be noted that most of the compounds,
especially those that are considerably active, were significantly
less toxic toward normal cells, when compared to cancer cells,
suggesting that the Xen analogues show selective toxic.
Cisplatin, a well-known anticancer agent, was used as a
positive control in our experiments and Xen were more
selective compared to cisplatin on normal cells. Finally, the
results suggest that the stereochemistry of Xens could possibly
play a vital role in the enhancement of bioactivity. Therefore,
finding the target binding site should reveal more information
about the connection between chiral properties and
bioactivity of Xens in the future.35, 36
(phenethylamino)-3-phenylpropan-2-yl)pentanamide (5)
The compound was obtained as a white solid; yield: (185.01
mg, 27%); [α] = -57 (c 0.2, MeOH); lit.13 = -54 (
c 0.2, MeOH);
ͦͤ
͸
1H NMR (600 MHz, CD3OD) showed the presence of two
rotamers in a ratio of 4.3/1; major rotamer noted: 7.29-7.24
(m, 6H, aryl), 7.21-7.17 (m, 4H, aryl), 5.42-5.39 (dd, J1 = 6.0 Hz,
J2 = 12.0 Hz, 1H, CH), 3.43-3.41 (t, = 7.2 Hz, 2H, CH2), 3.38-
3.36 (m, 1H, CH), 3.18-3.03 (dd, J1 = 5.4 Hz, J2= 14.4 Hz, 1H,
CH), 3.11-3.00 (m, 1H, CH ), 2.97 (s, 3H, CH3-N), 2.78-2.76 (t,
δ
J
J
=
7.2 Hz, 2H, CH2), 1.74 (s, 3H, CH3-NH), 1.30-1.25 (m, 2H, CH2),
1.15-1.14 (m, 1H, CH), 0.92-0.89 (m, 6H, 2CH3); 13C NMR (150
MHz, CD3OD) major rotamer noted:
δ 175.84, 170.75, 138.97,
137.13, 128.87, 128.74, 128.51, 128.14, 126.34, 126.01, 58.01,
57.57, 42.04, 40.46, 35.04, 34.22, 32.72, 30.70, 24.24, 22.49,
20.97 ppm; HRMS (TOF-ESI): [M+H]+calculated for C25H36N3O2:
410.2808; found: 410.2807.
Experimental
Methods and Materials
Chemistry
All reagents and solvents purchased were used without further (S)-N,4-dimethyl-2-(methylamino)-N-((R)-1-oxo-1-
purification unless otherwise stated. Reaction progress was
monitored using thin layer chromatography (TLC). TLC was
visualized using UV. Column chromatography was performed
using Whatman Purasil 60 A (230–400 mesh ASTM) silica gel;
yields refer to chromatographically and spectroscopically pure
compounds. 1H and 13C NMR were recorded using a Bruker
Avance III 600 spectrometer at 22 °C (default) unless otherwise
noted. The residual solvent signal in CD3OD was referenced to
3.31 and 49.0 ppm in proton and carbon spectra respectively.
Signal patterns are indicated as s: singlet; d: doublet; t: triplet;
q: quartet; m: multiplet; dd: doublet of doublets; br: broad and
coupling constants are reported in hertz (Hz). Low resolution
mass spectra (LRMS) were acquired on an Esquire-LC
(phenethylamino)-3-phenylpropan-2-yl)pentanamide (5a)
The compound was obtained as a white solid; yield: (127.59
mg, 18%); [α] = +43 (c
0.2, MeOH); 1H NMR (600 MHz,
ͦͤ
͸
CD3OD); δ 7.28-7.20 (m, 10H, aryl), 5.54-5.51 (dd, J1 = 6.0 Hz, J2
= 12.0 Hz, 1H, aryl), 3.77-3.75 (m, 1H, CH), 3.48-3.44 (m, 2H,
CH2), 3.31-3.25 (dd, J1 = 6.0 Hz, J2 = 12.0 Hz, 1H, CH2), 3.00-2.92
(m, 4H, CH, CH3-N), 2.82-2.80 (t,
3H, CH3-NH), 1.10-1.07 (m, 2H, CH2), 0.89- 0.85 (m, 1H, CH),
0.74-0.73 (d, = 6.0 Hz, 3H, CH3), 0.69-0.68 (d, = 6.0 Hz, 3H,
CH3); 13C NMR (150 MHz, CD3OD) major rotamer noted:
J = 6.0 Hz, 2H, CH2), 2.37 (s,
J
J
δ
174.85, 170.78, 138.98, 136.87, 128.49, 128.45, 128.20,
128.13, 126.55, 126.02, 57.45, 57.21, 41.03, 40.56, 35.01,
34.35, 32.61, 30.46, 23.83, 21.92, 21.33 ppm; HRMS (TOF-ESI):
[M+H]+calculated for C25H36N3O2: 410.2808; found: 410.2820.
electrospray ionization (ESI) mass spectrometer.
High
resolution mass spectra (HRMS) were obtained with a Bruker
Maxis 4G mass spectrometer.
(R)-N,4-dimethyl-2-(methylamino)-N-((R)-1-oxo-1-
(phenethylamino)-3-phenylpropan-2-yl)pentanamide (5b)
General Procedure
The compound was obtained as a white solid; yield: (102.30
mg, 15%); [α] = +57 (c 0.2, MeOH); 1H NMR (600 MHz,
ͦͤ
͸
To 0.27 mL (1.66 mmol) of methylamine (33% in ethanol) (
15 mL methanol, was added 0.18 mL (1.66 mmol) phenyl
acetaldehyde ( ), and the reaction was allowed to stir for 5
3) in
CD3OD) showed the presence of two rotamers in a ratio of
3.5/1; major rotamer noted:
7.16 (m, 4H, aryl), 5.43-5.40 (dd, J1 = 6.0 Hz, J2 = 12.0 Hz,1H,
CH), 3.44-3.36 (t, = 7.2 Hz, 2H, CH2), 3.35-3.33 (m, 1H), 3.20-
3.17 (dd, J1 = 5.2 Hz, J2= 14.3 Hz, 1H), 3.01-2.96 (m, 4H, CH,
CH3-N), 2.78-2.76 (t, = 7.2 Hz, 2H, CH2), 1.73 (s, 3H, CH3-NH),
1.29-1.24 (m, 2H, CH2), 1.14-1.13 (m, 1H, CH), 0.91-0.88 (m,
6H, 2CH3); 13C NMR (150 MHz, CD3OD) major rotamer noted:
δ 7.28-7.21 (m, 6H, aryl), 7.19-
2
minutes at room temperature.
Then, 1.66 mmol of
, and 1.66 mmol of
J
corresponding carboxylic acid 4a-b
isocyanide 1a-b were added sequentially and the reaction was
continuously stirred until no noticeable starting reagents were
visualized on TLC. Upon completion of the reaction, methanol
was evaporated under reduced pressure. After obtaining mass
of the unpurified Boc-protected product, the material was
dissolved in CH2Cl2 (1 mL) and trifluoroacetic acid (1 mL) was
added as the reaction mixture was allowed to chill in an ice-
J
δ
176.00, 170.75, 139.04, 137.48, 128.88, 128.74, 128.52,
126.60, 126.34, 126.07, 58.01, 57.99, 56.22, 40.47, 35.09,
34.24, 32.80, 30.71, 24.24, 21.95, 21.01 ppm; HRMS (TOF-ESI):
[M+H]+calculated for C25H36N3O2: 410.2808; found: 410.2823.
8 | J. Name., 2018, 00, 1-13
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ARTICLE
34.13, 30.34, 30.25, 24.63, 24.34, 22.47, 21.42 ppm; HRMS
(TOF-ESI): [M+H]+ calculated for C27H37N4O2: 449.2917 found
449.2931.
(R)-N,4-dimethyl-2-(methylamino)-N-((S)-1-oxo-1-
(phenethylamino)-3-phenylpropan-2-yl)pentanamide (5c)
The compound was obtained as a white solid; yield: (133.02
mg, 19%); [α] = -43 (c
0.2, MeOH); 1H NMR (600 MHz,
ͦͤ
͸
(R)-N-((R)-1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-
phenylpropan-2-yl)-N,4-dimethyl-2-
CD3OD);
δ = 7.29-7.17 (m, 10H, aryl), 5.55-5.52 (dd, J1 = 5.3 Hz,
(methylamino)pentanamide (6b)
J2 = 12.0 Hz, 1H, CH), 3.46-3.40 (m, 2H, CH2), 3.39-3.38 (m, 1H,
CH), 3.25-3.22 (dd, J1 = 5.3 Hz, J2 = 12.0 Hz, 1H, CH), 2.98-2.93
(m, 4H, CH, CH3-N), 2.81-2.78 (m, 2H, CH2), 2.20 (s, 3H, CH3-
NH), 0.99-0.95 (m, 1H, CH), 0.83-0.78 (m, 1H, CH), 0.77-0.74
The compound was obtained as a yellowish solid; yield:
(151.63 mg, 20%); [α] = +56 (c
0.3, MeOH); 1H NMR (600
ͦͤ
͸
MHz, CD3OD) showed the presence of two rotamers in a ratio
of 2.7/1; major rotamer noted: 7.56-7.55 (m, 1H, aryl), 7.33-
7.17 (m, 4H, aryl), 7.09-7.00 (m, 5H, aryl), 5.41-5.39 (dd, J1
(m, 1H, CH), 0.75 (d,
J
= 6.0 Hz, 3H, CH3), 0.66 (d,
J = 6.0 Hz, 3H,
δ
CH3) ppm; 13C NMR (150 MHz, CD3OD):
δ
176.43, 170.89,
=
138.99, 136.95, 128.49, 128.47, 128.21, 128.16, 126.47,
126.01, 57.36, 57.19, 41.74, 40.53, 35.03, 34.32, 33.11, 30.41,
23.91, 21.99, 21.49 ppm; HRMS (TOF-ESI): [M+H]+calculated
for C25H36N3O2: 410.2808; found: 410.2816.
5.6, J2 = 11.0 Hz, 1H, CH), 3.52-3.50 (m, 2H, CH2), 3.34-3.32 (m,
1H, CH), 3.19-3.15 (dd, J1 = 5.6 Hz, J2 = 14.4 Hz, 1H, CH), 3.00-
2.91 (m, 6H, CH, CH2, CH3-N), 1.69 (s, 3H, CH3-NH), 1.29-1.20
(m, 2H), 1.07-1.06 (m, 1H, CH), 0.86-0.84 (m, 6H, 2CH3) ppm;
13C NMR (150 MHz, CD3OD) major rotamer noted:
δ 176.03,
(S)-N-((S)-1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-
phenylpropan-2-yl)-N,4-dimethyl-2-
170.71, 137.19, 128.85, 128.42, 128.12, 127.38, 126.31,
122.07, 120.95, 118.23, 117.87, 110.96, 110.89, 61.43, 57.57,
43.08, 39.83, 34.08, 32.74, 30.63, 24.77, 24.27, 22.42, 20.99
ppm; HRMS (TOF-ESI): [M+H]+ calculated for C27H37N4O2:
449.2917 found 449.2914.
(methylamino)pentanamide (6)
The compound was obtained as a yellowish solid; yield:
(215.77 mg, 28%); [α] = -56 (c 0.3, MeOH); lit.13 -58 (c 0.3,
ͦͤ
͸
MeOH); 1H NMR (600 MHz, CD3OD) showed the presence of
(R)-N-((S)-1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-
phenylpropan-2-yl)-N,4-dimethyl-2-
two rotamers in a ratio of 2.8/1; major rotamer noted:
7.55 (d, = 6.0 Hz, 1H, aryl), 7.33-7.32 (d, = 6.0 Hz, 1H, aryl),
7.26-7.23 (m, 4H, aryl), 7.19-7.16 (m, 1H, aryl), 7.08-7.06 (t,
δ 7.56-
J
J
(methylamino)pentanamide (6c)
J
=
7.2 Hz, 1H, aryl), 7.04-6.99 (m, 2H, aryl), 5.40-5.39 (dd, J1 = 6.0,
J2 = 12.0 Hz, 1H, CH), 3.57-3.50 (m, 2H, CH2), 3.36-3.35 (m, 1H,
CH), 3.17-3.15 (dd, J1 = 5.4 Hz, J2 = 14.4 Hz, 1H, CH), 3.01-2.95
(m, 6H, CH, CH2, CH3-N), 1.73 (s, 3H, CH3-NH), 1.37-1.21 (m, 2H,
CH2), 1.07-1.06 (m, 1H, CH), 0.98-0.84 (m, 6H, 2CH3) ppm; 13C
The compound was obtained as a yellowish solid; yield:
(212.30 mg, 28%); [α] = -26 (c 0.3, MeOH); 1H NMR (600 MHz,
ͦͤ
͸
CD3OD) showed the presence of two rotamers in a ratio of
6.0/1; major rotamer noted:
aryl), 7.33-7.31 (d, = 7.8 Hz, 1H, aryl), 7.25-7.17 (m, 5H, aryl),
7.10-7.07 (m, 2H, aryl), 7.02-7.01 (t, = 7.8 Hz, 1H, aryl), 5.54-
δ = 7.57-7.56 (d, J = 7.8 Hz, 1H,
J
NMR (150 MHz, CD3OD) major rotamer noted:
δ 175.72,
J
170.68, 136.78, 128.74, 128.43, 128.13, 127.39, 126.32,
121.05, 120.96, 118.23, 117.88, 111.61, 110.89, 58.17, 57.56,
39.84, 39.77, 32.67, 30.77, 30.64, 24.77, 24.26, 22.41, 20.98
ppm; HRMS (TOF-ESI): [M+H]+calculated for C27H37N4O2:
449.2917 found 449.2933.
5.52 (dd, J1 = 4.32 Hz, J2 = 11.0 Hz, 1H, CH), 3.54-3.36 (m, 2H,
CH2), 3.35-3.31 (m, 1H), 3.23-3.20 (dd, J1 = 5.4, J2 = 15.0 Hz, 1H,
CH), 2.99-2.94 (m, 3H, CH, CH2), 2.88 (s, 3H, CH3-N), 2.21 (s, 3H,
CH3-NH), 0.96-0.93 (m, 2H, CH2), 0.84-0.79 (m, 1H, CH), 0.80-
0.69 (d, J = 6.0 Hz, 3H, CH3), 0.65-0.64 (d, J = 6.0 Hz, 3H, CH3)
ppm; 13C NMR (150 MHz, CD3OD) major rotamer noted:
δ
(S)-N-((R)-1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-
phenylpropan-2-yl)-N,4-dimethyl-2-
176.40, 170.82, 136.99, 136.78, 128.66, 128.14, 127.42,
126.44, 122.17, 120.95, 118.22, 117.90, 111.58, 110.85, 57.40,
57.15, 41.72, 39.84, 34.14, 32.98, 30.29, 24.63, 23.89, 21.96,
21.46; HRMS (TOF-ESI): [M+H]+ calculated for C27H37N4O2:
449.2917 found 449.2931.
(methylamino)pentanamide (6a)
The compound was obtained as a yellowish solid; yield:
(142.97 mg, 19%); [α] = +26° (c 0.3, MeOH); 1H NMR (600
ͦͤ
͸
MHz, CD3OD) showed the presence of two rotamers in a ratio
(S)-N,3-dimethyl-2-(methylamino)-N-((S)-1-oxo-1-
of 2.68/1; major rotamer noted:
aryl), 7.33-7.31 (d, = 7.8 Hz, 1H, aryl), 7.25-7.20 (m, 4H, aryl),
7.19-7.17 (t,
7.01 (t,
δ 7.57-7.56 (d, J = 7.8 Hz, 1H,
(phenethylamino)-3-phenylpropan-2-yl)butanamide (7)
J
J
= 7.2 Hz, 1H, aryl), 7.09-7.06 (m, 2H, aryl), 7.02- The compound was obtained as a white solid; yield: (166.99
ͦͤ
͸
J
= 7.8 Hz, 1H, aryl), 5.54-5.51 (dd, J1 = 5.4 Hz, J2 = 11.4 mg, 25%); [α] = -54 (c
0.2, MeOH); 1H NMR (600 MHz, CD3OD)
Hz, 1H, CH), 3.55-3.52 (m, 2H, CH2), 3.40-3.37 (m, 1H, CH), showed the presence of two rotamers in a ratio of 7.70/1;
3.17-3.15 (dd, J1 = 5.4, J2 = 15.0 Hz, 1H, CH), 2.99-2.94 (m, 3H, major rotamer noted: 7.29-7.27 (m, 6H, aryl), 7.22-7.7.19 (m,
CH, CH2), 2.88 (s, 3H, CH3-N), 2.21 (s, 3H, CH3-NH), 0.96-0.95 4H, aryl), 5.51-5.49 (dd, J1 = 5.4 Hz, J2 = 11.4 Hz, 1H, CH), 3.50-
(m, 2H, CH2), 0.84-0.79 (m, 1H, CH2), 0.80-0.69 (d, = 6.0 Hz, 3.44 (t, J =7.2 Hz, 2H, CH2), 3.22-2.99 (m, 6H, CH, CH2, CH3-N),
3H, CH3), 0.65-0.64 (d,
= 6.0 Hz, 3H, CH3) ppm; 13C NMR (150 2.07-2.04 (m, 1H, CH), 1.97 (s, 3H, CH3-NH), 1.04-1.03 (d,
MHz, CD3OD) major rotamer noted: 176.17, 170.78, 136.95, 6.6 Hz, 3H, CH3), 0.98-0.97 (d,
= 6.6 Hz, 3H, CH3) ppm; 13C
136.77, 128.65, 128.16, 127.42, 126.46, 122.17, 120.96, NMR (150 MHz, CD3OD) major rotamer noted: 170.13,
118.23, 117.91, 111.58, 110.86, 57.45, 57.19, 41.58, 39.87, 167.65, 138.92, 136.91, 128.46, 128.23, 128.18, 128.13,
δ
J
J
J =
δ
J
δ
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126.54, 126.03, 63.45, 58.06, 40.44, 34.98, 34.97, 34.05, 31.17, The compound was obtained as a yellowish solid; yield:
29.81, 17.67, 16.10 ppm; HRMS (TOF-ESI): [M+H]+calculated (142.02 mg, 20%); [α] = -58 ( 0.3, MeOH); 1H NMR (600 MHz,
c
ͦͤ
͸
for C24H34N3O2: 396.2651; found: 396.2645.
CD3OD) showed the presence of two rotamers in a ratio of
3.8/1; major rotamer noted: 7.55-7.54 (d, = 7.8 Hz, 1H,
aryl), 7.33-7.31 (d, = 7.8 Hz, 1H, aryl), 7.23-7.18 (m, 4H, aryl),
7.16-7.15 (m, 1H, aryl), 7.09-7.08 (t, = 7.2 Hz, 1H, aryl), 7.04
(s, 1H, aryl), 6.99 (t, = 7.2 Hz, 1H, aryl), 5.47 (dd, J1 = 6.0 Hz,
J2= 10.8 Hz, 1H, CH), 3.52-3.50 (m, 2H, CH2), 3.18-3.14 (m, 1H,
CH), 3.10-3.08 (d, = 6.0 Hz, 1H, CH), 2.99-2.91 (m, 6H, CH,
CH2, CH3-N), 1.68 (s, 3H, CH3-NH), 1.60-1.57 (m, 1H, CH), 0.84-
0.83 (d, = 6.6 Hz, 3H, CH3), 0.81 (d, = 6.6 Hz, 3H, CH3) ppm;
13C NMR (150 MHz, CD3OD) major rotamer noted:
175.35,
δ
J
(S)-N,3-dimethyl-2-(methylamino)-N-((R)-1-oxo-1-
J
(phenethylamino)-3-phenylpropan-2-yl)butanamide (7a)
J
J
The compound was obtained as a white solid; yield: (129.45
mg, 19%); [α] = +29 (c
0.2, MeOH); 1H NMR (600 MHz,
ͦͤ
͸
J
CD3OD) showed the presence of two rotamers in a ratio of
2.96/1; major rotamer noted: 7.29-7.26 (m, 6H, aryl), 7.22-
δ
7.20 (m, 4H, aryl), 5.55-5.49 (dd, J1 = 5.4 Hz, J2 = 12.0 Hz, 1H,
J
J
δ
CH), 3.50-3.46 (m, 2H, CH2), 3.27-3.23 (m, 2H, CH2), 3.02-2.98
170.66, 137.15, 136.80, 128.72, 128.35, 128.11, 126.30,
122.07, 120.95, 118.22, 117.87, 111.50, 110.86, 64.47, 57.65,
39.64, 34.03, 33.03, 30.93, 30.57, 24.79, 18.55, 16.40 ppm;
HRMS (TOF-ESI): [M+H]+calculated for C26H35N4O2: 435.2760;
found: 435.2754.
(m, 4H, CH, CH3-N), 2.83-2.81 (t,
3H, CH3-NH), 1.66-1.59 (m, 1H, CH), 0.75-0.74 (d,
CH3), 0.53-0.52 (d,
= 6.6 Hz, 3H, CH3) ppm; 13C NMR (150
MHz, CD3OD) major rotamer noted: 170.53, 170.19, 138.98,
J
= 7.2 Hz, 2H, CH2), 2.48 (s,
J
= 6.6 Hz, 3H,
J
δ
136.79, 128.52, 128.50, 128.34, 128.14, 126.60, 126.04, 63.97,
57.91, 40.59, 34.99, 34.28, 30.76, 29.51, 17.47, 15.66, 13.04
ppm; HRMS (TOF-ESI): [M+H]+calculated for C24H34N3O2:
396.2651; found: 396.2640.
(S)-N-((R)-1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-
phenylpropan-2-yl)-N,3-dimethyl-2-
(methylamino)butanamide (8a)
(R)-N,3-dimethyl-2-(methylamino)-N-((R)-1-oxo-1-
The compound was obtained as a white solid; yield: (109.67
mg, 15%); [α] = +28 (c
0.3, MeOH); 1H NMR (600 MHz,
ͦͤ
͸
(phenethylamino)-3-phenylpropan-2-yl)butanamide (7b)
The compound was obtained as a white solid; yield: (142.68
CD3OD) showed the presence of two rotamers in a ratio of
6.6/1; major rotamer noted: 7.56-7.55 (d, = 7.8 Hz, 1H,
aryl), 7.33-7.32 (d, = 7.8, 1H, aryl), 7.24-7.21 (m, 4H, aryl),
7.17-7.7.15 (m, 1H, aryl), 7.09-7.08 (t, = 7.2 Hz, 1H, aryl), 7.05
(s, 1H, aryl), 7.01-6.99 (t, = 7.2 Hz, 1H, aryl), 5.52-5.49 (dd, J1
= 5.4 Hz, J2= 11.4 Hz, 1H, CH), 3.53-3.51 (t, = 6.6 Hz, 2H, CH2),
3.22-3.18 (m, 2H, CH2), 2.96-2.90 (m, 6H, CH, CH2, CH3-N), 2.08
(s, 3H, CH3-NH), 1.36-1.29 (m, 1H, CH), 0.62-0.60 (d, = 6.6 Hz,
3H, CH3), 0.51-0.50 (d,
= 6.6 Hz, 3H, CH3) ppm; 13C NMR (150
MHz, CD3OD) major rotamer noted: 175.36, 170.85, 137.06,
mg, 21%); [α] = +54 (c
0.2, MeOH); 1H NMR (600 MHz,
ͦͤ
͸
δ
J
CD3OD) showed the presence of two rotamers in a ratio of
4.2/1; major rotamer noted: 7.29-7.23 (m, 6H, aryl), 7.20-
7.17 (m, 4H, aryl), 5.51 (d.d, J1 = 5.4, J2 = 10.8 Hz, 1H, CH), 3.44
(t, = 6.6 Hz, 2H, CH2), 3.22-3.21 (m, 2H, 2CH), 3.00-2.96 (m,
J
δ
J
J
J
J
4H, CH, CH3-N), 2.78-2.75 (m, 2H, CH2), 1.71-1.66 (m, 4H, CH,
CH3-NH), 0.92-0.88 (m, 6H, 2CH3); 13C NMR (150 MHz, CD3OD)
J
major rotamer noted:
δ 175.55, 170.46, 138.98, 137.03,
J
128.97, 128.63, 128.48, 128.19, 128.13, 126.38, 64.42, 57.52,
40.51, 35.03, 34.31, 33.65, 30.50, 30.28, 18.37, 16.24; HRMS
(TOF-ESI): [M+H]+ calculated for C24H34N3O2: 396.2651; found:
396.2644.
δ
136.78, 128.62, 128.17, 127.39, 126.36, 122.15, 120.96,
118.23, 117.89, 110.43, 110.86, 64.38, 57.60, 39.79, 33.38,
33.49, 30.49, 30.25, 24.66, 18.32, 16.21 ppm; HRMS (TOF-ESI):
[M+H]+calculated for C26H35N4O2: 435.2760 found 435.2775.
(R)-N,3-dimethyl-2-(methylamino)-N-((S)-1-oxo-1-
(R)-N-((R)-1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-
phenylpropan-2-yl)-N,3-dimethyl-2-
(phenethylamino)-3-phenylpropan-2-yl)butanamide (7c)
(methylamino)butanamide (8b)
The compound was obtained as a white solid; yield: (156.95
mg, 23%); [α] = -29 (c
0.2, MeOH); 1H NMR (600 MHz, CD3OD)
ͦͤ
͸
The compound was obtained as a yellowish solid; yield:
(121.13 mg, 17%); [α] = +58 (c
0.3, MeOH); 1H NMR (600
ͦͤ
͸
showed the presence of two rotamers in a ratio of 6.3/1;
major rotamer noted: 7.29-7.23 (m, 7H, aryl), 7.20-7.17 (m,
3H, aryl), 5.51 (d.d, J1 = 5.4, J2 = 10.8 Hz. 1H, CH), 3.44 (t, = 6.6
δ
MHz, CD3OD) showed the presence of two rotamers in a ratio
of 3.2/1; major rotamer noted: 7.55 (d, = 7.8 Hz, 1H, aryl),
7.32 (d, = 7.8 Hz, 1H, aryl), 7.24-7.22 (m, 1H, aryl), 7.18-7.15
(m, 1H, aryl), 7.08 (t, = 7.8 Hz, 1H, aryl), 7.04-7.00 (m, 1H,
J
δ
J
Hz, 2H, CH2), 3.22-3.21 (m, 2H, CH2), 2.99-2.93 (m, 4H, CH, CH3-
N), 2.83-2.77 (m, 2H, CH2), 2.17 (s, 3H, CH3-NH), 1.40-1.36 (1H,
J
J
m, CH), 0.63 (d, J = 6.6 Hz, 3H, CH3), 0.53 (d, J = 6.6 Hz, 3H,
CH3); 13C NMR (150 MHz, CD3OD) major rotamer noted:
aryl), 5.48 (d.d, J1 = 5.4, J2 = 11.4 Hz, 1H, CH), 3.53-3.50 (m, 2H,
δ
CH2), 3.17-3.12 (m, 2H, CH2), 2.99-2.92 (m, 6H, CH, CH2, CH3-
175.55, 170.46, 138.98, 137.03, 128.97, 128.63, 128.48,
128.19, 128.13, 126.38, 64.42, 57.52, 40.51, 35.03, 34.31,
33.65, 30.50, 30.28, 18.37, 16.24; HRMS (TOF-ESI): [M+H]+
calculated for C24H34N3O2: 396.2651; found: 396.2644.
N), 1.70 (s, 3H, CH3-NH), 1.62-1.58 (m, 1H, CH), 0.90 (d,
Hz, 3H, CH3), 0.82 (d,
= 7.2 Hz, 3H, CH3); 13C NMR (150 MHz,
CD3OD) major rotamer noted: 171.43, 170.64, 137.15,
J = 7.2
J
δ
136.80, 128.73, 128.11, 127.37, 126.30, 121.05, 120.95,
118.22, 117.87, 111.51, 110.86, 64.45, 57.66, 39.64, 34.09,
32.99, 30.89, 30.58, 24.79, 18.57, 16.67; HRMS (TOF-ESI):
[M+H]+calculated for C26H35N4O2: 435.2760; found: 435.2755.
(S)-N-((S)-1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-
phenylpropan-2-yl)-N,3-dimethyl-2-
(methylamino)butanamide (8)
10 | J. Name., 2018, 00, 1-13
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experiment, various concentrations of each compound ranging
from 0.10 to 100.0 µM (20.0 µL/well) were added to the wells.
After 72 h of incubation, 20.0 µL of the MTT solution (4.0
mg/mL) was added to each well. The plates were further
incubated for 4 h to allow the viable cells to bio transform the
yellow-colored MTT into dark-blue formazan crystals. The
medium was discarded and 100.0 µL of DMSO was used to
dissolve the formazan crystals. The absorbance was measured
at 570 nm using the SpectraMax® iD3 H1 Multi-Mode Micro
Reader from Molecular Devices (Sunnyvale, CA, USA). The IC50
values were calculated from the cell survival percentages.
Similarly, the cytotoxicity of the test compounds was
compared to normal cell lines CHO and CRL-1459.
(R-N-((S)-1-((2-(1H-indol-3-yl)ethyl)amino)-1-oxo-3-phenylpropan-
2-yl)-N,3-dimethyl-2-(methylamino)butanamide (8c)
The compound was obtained as a white solid; yield: (182.30
mg, 25%); [α] = -28 (c
0.3, MeOH); 1H NMR (600 MHz, CD3OD)
ͦͤ
͸
showed the presence of two rotamers in a ratio of 5.9/1;
major rotamer noted: 7.56-7.54 (d, = 7.8 Hz, 1H, aryl), 7.33-
7.31 (d, = 7.8 Hz, 1H, aryl), 7.22-7.20 (m, 4H, aryl), 7.17-7.15
(m, 1H, aryl), 7.08-7.07 (t, = 7.8 Hz, 1H, aryl), 7.05 (s, 1H, aryl),
7.02-7.01 (m, 1H, aryl), 5.52-5.49 (d.d, J1 = 6.0, J2 = 12.0 Hz, 1H,
CH), 3.53-3.51 (t, = 7.2 Hz, 2H, CH2), 3.21-3.18 (d.d, J1 = 3.6, J2
= 15.0 Hz, 1H, CH), 3.15-3.14 (d, = 5.4, 1H, CH), 2.96-2.93 (m,
3H, CH, CH2), 2.88 (s, 3H, CH3-N), 2.07 (s, 3H, CH3-NH), 1.34-
1.33 (m, 1H, CH), 0.60 (d, = 6.6 Hz, 3H, CH3), 0.50 (d, = 6.6
Hz, 3H, CH3); 13C NMR (150 MHz, CD3OD) major rotamer
noted: 175.59, 170.86, 137.79, 128.63, 128.52, 128.17,
δ
J
J
J
J
J
J
J
Cell Morphological Analysis
Cell images were taken using Evos® FL cell imaging system
microscope from Thermo- Fisher Scientific (Waltham, MA)
with a fluorescent lamp and digital camera. Morphological
changes were observed in cancer and normal cell lines 72 h
after incubation with different concentrations of Xen
compounds.
δ
127.40, 126.35, 122.16, 120.97, 118.25, 117.91, 111.58,
110.88, 64.41, 57.60, 39.80, 34.13, 33.55, 30.44, 30.29, 24.68,
18.37, 16.25; HRMS (TOF-ESI): [M+H]+calculated for
C26H35N4O2: 435.2760; found: 435.2726.
Biology
Materials
Conflicts of interest
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) was purchased from Calbiochem EMD Millipore
(Billerica, MA, USA). Dulbecco’s modification of Eagle’s
There are no conflicts of interest to declare.
medium (DMEM) and 0.25% trypsin
+
2.20 mM
Acknowledgements
ethylenediaminetetraacetic acid (EDTA), phosphate buffered
saline (PBS without calcium or magnesium), and DMEM The authors gratefully acknowledge the University of Toledo
(phenol red - free) were purchased from Mediatech, Inc. for generous support of this project.
(Corning subsidiaries, Manassas, VA, USA). Fetal Bovine Serum
(FBS) was purchased from Atlanta Biologicals (Flowery Branch,
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