Anticancer activity of small amphipathic β2,2-amino acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.09.048

We report the anticancer activity from screening of a series of synthetic β^2,2-amino acid derivatives that were prepared to confirm the pharmacophore model of short cationic antimicrobial peptides with high anti-Staphylococcal activity. The most potent derivatives against human Burkitt's lymphoma (Ramos) cells displayed IC₅₀ values below 8 μM, and low toxicity against human red blood cells (EC₅₀ > 200 μM). A more than 5-fold preference for Ramos cancer cells compared to human lung fibroblasts (MRC-5 cells) was also obtained for the most promising β^2,2-amino acid derivative 3-amino-N-(2-aminoethyl)-2,2- bis(naphthalen-2-ylmethyl)propanamide (5c). Screening of 5c at the National Cancer Institute (NCI, USA) confirmed its anticancer potency and revealed a very broad range of anticancer activity with IC₅₀ values of 0.32-3.89 μM against 59 different cancer cell lines. Highest potency was obtained against the colon cancer cell lines, a non-small cell lung cancer, a melanoma, and three leukemia cell lines included in the NCI screening panel. The reported β^2,2-amino acid derivatives constitute a promising new class of anticancer agents based on their high anticancer potency, ease of synthesis, mode-of-action, and optimized pharmacokinetic properties compared to much larger antimicrobial peptides.

Full text of DOI:10.1016/j.ejmech.2012.09.048

European Journal of Medicinal Chemistry 58 (2012) 22e29
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anticancer activity of small amphipathic b
2,2-amino acid derivatives
,
Terkel Hansen a b, Dominik Ausbacher a, Zack G. Zachariassen a, Trude Anderssen a, Martina Havelkova a,
,
Morten B. Strøm a
*
a Department of Pharmacy, Faculty of Health Sciences, University of Tromsø, NO-9037 Tromsø, Norway
b Leibniz-Institut für Analytische Wissenschaften e ISAS e e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany
a r t i c l e i n f o
a b s t r a c t
We report the anticancer activity from screening of a series of synthetic b
2,2-amino acid derivatives that
were prepared to confirm the pharmacophore model of short cationic antimicrobial peptides with high
anti-Staphylococcal activity. The most potent derivatives against human Burkitt’s lymphoma (Ramos)
Article history:
Received 17 April 2012
Received in revised form
17 September 2012
Accepted 28 September 2012
Available online 5 October 2012
cells displayed IC50 values below 8
more than 5-fold preference for Ramos cancer cells compared to human lung fibroblasts (MRC-5 cells)
was also obtained for the most promising
2,2-amino acid derivative 3-amino-N-(2-aminoethyl)-2,2-
mM, and low toxicity against human red blood cells (EC50 > 200 mM). A
b
bis(naphthalen-2-ylmethyl)propanamide (5c). Screening of 5c at the National Cancer Institute (NCI,
USA) confirmed its anticancer potency and revealed a very broad range of anticancer activity with IC50
Keywords:
Anticancer peptide
Antimicrobial peptide
Amphipathic scaffold
Beta-amino acid
Peptidomimetics
values of 0.32e3.89
colon cancer cell lines, a non-small cell lung cancer, a melanoma, and three leukemia cell lines included
in the NCI screening panel. The reported
2,2-amino acid derivatives constitute a promising new class of
mM against 59 different cancer cell lines. Highest potency was obtained against the
b
anticancer agents based on their high anticancer potency, ease of synthesis, mode-of-action, and opti-
mized pharmacokinetic properties compared to much larger antimicrobial peptides.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Intracellular targets have also been identified for anticancer
AMPs in vitro [15]. Studies have shown that lactoferricin B (LfcinB)
Cationic antimicrobial peptides (AMPs) are a diverse group of
peptides contributing to the innate immunity of mammals,
amphibians and insects [1e3]. Excitingly, reports have also shown
that the cationic and amphipathic nature of AMPs can be exploited
to design AMP-based peptides and peptidomimetics that also kill
cancer cells [4e7]. Anticancer AMPs exert a unique mode-of-action
compared to traditional anticancer drugs by preferring binding to
cancer cells with elevated levels of anionic surface molecules, such
as phosphatidylserine, O-glycosylated mucins, and sialylated
glycolipids and glycoproteins, followed by rapid lysis of the cancer
cell membrane [4,8e14].
translocates across the cancer cell membrane and acts by disrupt-
ing the mitochondrial membrane, as observed by swelling of the
mitochondria and induction of apoptosis [16]. Furthermore, in vivo
studies have revealed that mice treated with a synthetic LfcinB
derived peptide became resistant to reintroduction of the same
cancer cell line, implying that the membranolytic mechanism of
action of the anticancer AMPs may activate adaptive immunity
against established tumors [17,18].
Although cancer cells are notorious for developing resistance
against chemotherapeutic drugs, e.g. by increased expression of
efflux pumps or mutations in key regulatory proteins, studies have
shown that multi-drug resistant cancer cells are still susceptible to
anticancer AMPs due to their membrane lytic mechanism of action
[5,19,20]. Anticancer AMPs are therefore a promising class of
molecules for future cancer therapy, either as therapeutic agents
alone, or in combination therapy with traditional chemothera-
peutic drugs against resistant tumors [11,20].
Abbreviations: AMP, cationic antimicrobial peptide; BOC2O, di-tert-butyl dicar-
bonate; DIPEA, di-isopropyl ethyl amine; 5-FU, 5-fluorouracil; LfcinB, bovine lac-
toferricin; MBC, minimum bactericidal concentration; MIC, minimum inhibitory
concentration; MD, molecular dynamics; MRC-5, human lung fibroblasts; MRSA,
methicillin resistant Staphylococcus aureus; MRSE, methicillin resistant Staphylo-
coccus epidermidis; NCI, National Cancer Institute; PBMCs, peripheral blood
mononuclear cells; RBC, human red blood cells; TEA, triethyl amine; TFFH, fluoro-
N,N,N0,N0-tetramethylformamidinium hexafluorophosphate; TMRE, tetrame-
thylrhodamine ethyl ester; SI, selectivity index.
We have recently demonstrated that the minimal pharmaco-
phore elements of small AMPs, i.e., amphipathic peptides enclosing
two bulky lipophilic groups in combination with a net charge of þ2,
can be incorporated into a single
resulting
b
2,2-amino acid derivative. The
-peptidomimetics display high antimicrobial activity
* Corresponding author. Tel.: þ47 77 64 40 85; fax: þ47 77 64 61 51.
b
E-mail address: morten.strom@uit.no (M.B. Strøm).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.048

T. Hansen et al. / European Journal of Medicinal Chemistry 58 (2012) 22e29
23
against methicillin resistant Staphylococcus aureus (MRSA) and
methicillin resistant Staphylococcus epidermidis (MRSE), Escherichia
coli, and also Pseudomonas aeruginosa for the overall most potent
derivatives [21e23].
made an important alteration of the reported synthesis of the
precursors 1aec by replacing sodium methoxide with potassium
carbonate as base and stirring 2 eq. of the desired aryl bromide for
18 h (method a2 in Scheme 1), in comparison to two sequential
reactions with sodium methoxide and 1 eq. aryl bromide (method
a1 in Scheme 1). These adjustments were the key to both the
improved crude purity and overall yields of 9aec (see the
Supporting information for details).
Compared with per orally administered commercial drugs the
absorption properties of the
than satisfactory both in theoretical calculations and in vitro studies
[22]. The
2,2-amino acid derivatives are also highly stable against
b
2,2-amino acid derivatives were more
b
proteolytic degradation, but are metabolized through CYP450
Phase I oxidations, and thereby follow common metabolic routes of
many commercial drugs [24].
2.2. Anticancer activity
Recently, we have reported that anticancer activity can be
The results revealed that all the prepared b
2,2-amino acid
achieved by using these
blocks in small synthetic peptides and shown how structural
differences of the
2,2-amino acids affect anticancer potency and
toxicity against non-malignant cells [6,7]. The results show that
these small peptides containing a
2,2-amino acid building block
b
2,2-amino acids as lipophilic building
derivatives displayed anticancer activity against the Ramos cells,
except for one, and that nine different derivatives displayed IC50
b
values below 8 mM (Table 1). The present b
2,2-amino acid deriva-
tives are thereby amongst the smallest anticancer AMP-based
peptidomimetics reported together with the lipopeptides
patented by the research group of Shai and our own reported small
synthetic peptides [5e7,25e28].
b
with two (a) tert-butyl-benzyl, (b) para-trifluoromethyl-benzyl, or
(c) 2-napthyl-methylene aromatic side-chains are highly potent.
Importantly, one of the most promising peptides is a heptapeptide
which is non-toxic against human red blood cells (RBCs) and
peripheral blood mononuclear cells (PBMCs), and also shows low
toxicity against human lung fibroblasts (MRC-5 cells) [6].
Among the most potent compounds, five were b
2,2-amino acid
derivatives containing two lipophilic 2-napthyl-methylene side-
chains (5c, 6c, 9c, 10c, 11c), which was clearly an efficient motif for
anticancer activity. Also 11a and 12a having two tert-butyl-benzyl
side-chains, and 5b and 9b comprising two para-trifluoromethyl-
benzyl side-chains were highly potent and displayed IC50 values
Through the discovery of anticancer activity of the small
synthetic peptides, we have in the present study investigated if
single
b
2,2-amino acid derivatives enclosing the three most potent
below 8 mM. It can be noted that all derivatives except for 7aec
aromatic side-chains (a)e(c) from the larger synthetic peptides
contained an N-terminal primary amino group, and all derivatives
were expected to have a net charge of þ2 at the test conditions used.
However, by observing the variation in anticancer potency and side-
chain structure, it was obvious that the potency also was influenced
by the physicochemical nature of the cationic groups.
would display anticancer activity. We have previously shown how
the C-terminal cationic group of such b
2,2-amino acid derivatives
can tune both antimicrobial activity and cell toxicity [22]. In order
to investigate the structural importance of the cationic groups,
eight different N- and/or C-terminal amino and guanidinium
groups were therefore selected in the present study that in total
The overall most potent b
2,2-amino acid derivatives were 9b and
9c, which had a C-terminal tertiary amino group. However, 9b and
9c were also unique by being ester derivatives. Although it is
difficult to determine the specific contribution of the ester group
due to small differences in potency, the somewhat higher rotational
freedom and flexibility of the ester derivatives 9b and 9c could be
favorable with respect to cancer cell interactions, and compared to
the double bond character of the bioisosteric amide derivatives 10b
and 10c. The results nevertheless suggest the possibility of
replacing both the ester and amide functionalities with other
functional groups in order to further optimize the derivatives. It
should however be noted that the ester derivate 9a encompassing
two tert-butyl-benzyl side-chains displayed similar potency against
the Ramos cell line as the amide derivative 10a. A C-terminal
primary amino group (5aec and 6aec) seemed furthermore to be
more efficient than a C-terminal tertiary amino group (10aec). Of
note, the only difference between the two series 5aec and 6aec
was a single methylene group, and a small effect favoring the
smaller derivatives 5aec was also observed.
formed a test series of 24 different
(Fig. 1).
The
activity against human Burkitt’s lymphoma (Ramos cells), and
toxicity against human RBCs and MRC-5 cells. The current b2,2
b
2,2-amino acid derivatives
b
2,2-amino acid derivatives were screened for anticancer
-
amino acid derivatives are to our knowledge among the smallest
anticancer AMP-based peptidomimetics reported, and the anti-
cancer activity of the most promising derivative 5c was also
confirmed by screening at the National Cancer Institute (NCI, USA)
revealing high potency against 59 different cancer cell lines.
2. Results and discussion
2.1. Synthesis
Synthesis of the dicationic
10e12 was carried out as previously reported in a four step
synthesis by first preparing the three Boc-protected
2,2-amino
acids 3aec (Scheme 1) [21,22]. Due to high steric hindrance of the
2,2-amino acids, the following amide couplings were performed
b
2,2-amino acid derivatives 5e8 and
b
We have recently reported a detailed mode-of-action study on 5c
that suggests that b
2,2-amino acid derivatives might not kill cancer
b
cells exclusively by a membrane lytic mechanism [29]. In fact,
incubation of Ramos cells with 5c initiates controlled cell death
involving the mitochondrial apoptotic pathway, and flow cytometry
studies with tetramethylrhodamine ethyl ester (TMRE) show that 5c
is able to disrupt the mitochondrial potential of Ramos cancer cells
within 1 h. Further investigations for hallmarks of apoptosis and
electron microscopy studies were consistent with these findings.
Previous results have also shown that 5c is able to cross mimics of
using TFFH as coupling reagent to give the final test series 5e8 and
10e12.
The dicationic ester derivatives 9aec were on the other hand
prepared by transesterification of the methyl ester of the precursors
1aec with 2-(dimethylamino)ethanol, followed by reduction of the
nitrile group in 4aec (Supporting information). Synthesis of 9aec
gave overall higher purity and yields of the crude products
compared to the dicationic amide derivatives 5e8 and 10e12. The
major contaminant in the crude products of 9aec was approxi-
mately 10% of the non-hydrogenated nitriles 4aec (based on HPLC-
UV chromatograms), which probably can be reduced by more
careful monitoring of the reduction of the nitrile group. We also
biological membranes by passive diffusion [22]. A different b2,2
-
amino acid derivative with two phenyl-propyl side-chains did,
however, not act by a similar mechanism as demonstrated for 5c,
but led to necrotic cell death by membrane destabilization as
observed by increased propidium iodide uptake [29].

Fig. 1. Structure of the antimicrobial b
2,2-amino acid derivatives that were prepared for investigation of anticancer activity against human Burkitt’s lymphoma (Ramos). Derivative
5c was also subjected to an extensive screening against 59 cancer cell lines at the National Cancer Institute (NCI). All the
trifluoroacetate salts.
b
2,2-amino acid derivatives were isolated as di-

T. Hansen et al. / European Journal of Medicinal Chemistry 58 (2012) 22e29
25
O
O
R
O
R
O
R
N
N
N
b)
i)
a1)
HN
N
O
O
O
H3N
O
or a2)
R
R
R
1a-c
4a-c
9a-c
b) c)
O
O
R
O
R
Boc
Boc
N
O
HN
NH3
R
R
2a-c
H
H3N
H3N
N
H
H3N
N
H
R
R
5a-c
10a-c
d)
O
O
O
R
H3N
N
N
N
R
R
11a-c
H
N
NH3
N
OH
N
R
R
H
R
H
e) f) g)
e) f) g)
6a-c
3a-c
O
h)
e) f) g)
H3N
R:
R
R
12a-c
NH
H2N
NH2
NH
NH2
O
NH2
NH2
a
b
c
H2N
N
H
N
N
R
R
H
H
O
R
NH2
7a-c
H3N
N
H
R
O
CF3
8a-c
Scheme 1. Overview of the b2,2-amino acid derivatives that were synthesized for investigation of anticancer activity. (a1) NaOMe (1 eq), ReBr (1 eq), performed twice, 78 ꢀC s:
MeOH (a2) K2CO3 (3 eq), ReBr (2 eq), 50 ꢀC, 18 h, s: DMF (b) Ra/Ni, H2 (g), 45 ꢀC, 5 days, s: MeOH. (c) TEA, pH 8, Boc2O (1.2 eq), rt, 18 h, s: H2O:dioxane (1:5). (d) LiOH (6 eq), 18 h,
100 ꢀC, s: H2O:dioxane (1:3). (e) DIPEA (3 eq), TFFH (1.2 eq), rt, s: DMF. (f) HeArgeNH2 ꢁ 2 HCl or desired amine (2 eq), DIPEA, rt, 7 days, s: DMF. (g) TFA:TIS:H2O (95:2.5:2.5), rt 2 h,
s: DCM. (h) DIPEA (8 eq), 1H-1,2,4-triazole-1-carboxamidine ꢁ HCl (5 eq), r.t. 2 h, s: DMF, (i) 0.1 eq NaOMe, s: N,N-dimethyl 2-aminoethanol 50 ꢀC, 18 h.
The recent studies on 5c may also explain the surprisingly low
potency of the guanidinium derivatives 7aec and 8aec. Our
previous investigations of antimicrobial activity have revealed
Small differences in the structures of the b
2,2-amino acid
derivatives therefore seemed to have a major impact on their
interactions with cancer cell membranes and their detailed mode-
of-action. With respect to the macroscopic physiochemical prop-
erties of the derivatives and their membrane interactions, we have
recently concluded a study on small synthetic linear and cyclic
similar potencies for amino and arginine b
2,2-amino acid deriva-
tives, but the present results showed that anticancer activity was
much more sensitive to such structural alterations [21,22]. The low
potency of the guanidinium derivatives was suspected to be the
result of a more dispersed cationic charge and thereby reduced
electrostatic interaction with the cancer cell membrane of the
guanidinium groups than for the primary and tertiary amino
groups [30,31]. However, another explanation may be that the
much higher pKa of the guanidinium groups may prevent 7aec and
8aec from interacting efficiently with the membrane and entering
the cancer cells through passive diffusion due to permanent ioni-
zation, and thereby preclude interaction with important intracel-
lular targets such as the mitochondria.
peptides containing a b
2,2-amino acid residue where these inter-
actions were studied through nuclear magnetic resonance (NMR)
spectroscopy and molecular dynamics (MD) simulations [7]. The
results show that adjacent a-amino acid residues and their overall
structures affected both the orientation and depth of peptide
insertion into the model membranes. Thus, in a linear synthetic
hexapeptide only one of the lipophilic b
2,2-amino acid side-chains
was in fact inserted into the bilayer together with an adjacent
lysine residue, which probably explained its low anticancer
potency. However, in a cyclic hexapeptide with the same amino
acid sequence but with an overall more amphipathic structure,
While rotational freedom was increased by the highly potent
ester derivatives 9bec, introduction of a piperazine ring in the
cationic C-terminal end restricted the flexibility of 11aec and 12ae
c. Of note, 11a, 11c, and 12a were still among the nine highly potent
both the lipophilic
b
2,2-amino acid side-chains together with
a nearby tryptophan residue were inserted deep into the bilayer
surface and resulted in a highly potent membranolytic peptide. (In
b
2,2-amino acid derivatives displaying anticancer potencies below
this particular study peptides with a
side-chains of structure b, para-trifluoromethyl-benzyl were used).
The present
2,2-amino acid derivatives are too small and flexible
b
2,2-amino acid having two
8
mM against Ramos cancer cells. When similar derivatives with
respect to side-chain structure were compared, the C-terminal
modified piperazine derivatives revealed a fairly higher anticancer
potency for the largest but more flexible derivatives 11aec,
compared to the smaller and more restricted analogs 12aec.
Furthermore, derivatives 11aec contained in theory three basic
amino groups, but due to probable charge repulsion between the C-
terminal basic amino groups, we expect these derivatives to be only
di-cationic and have a net charge of þ2.
b
for similar studies. However, the results from comparison of the
linear and cyclic hexapeptides imply that the overall macroscopic
physiochemical properties of the b
2,2-amino acid residues, i.e. steric
effects of the lipophilic side-chains, pKa and structure of the
cationic groups, amphipathicity and overall conformation have
a substantial influence on membrane activity. This affects how the
derivatives orient into a membrane, how deep they insert, and

26
T. Hansen et al. / European Journal of Medicinal Chemistry 58 (2012) 22e29
Table 1
Anticancer potency of
2.4. NCI screening of 5c
b
2,2-amino acid derivatives against human Burkitt’s
lymphoma (Ramos) cells, and toxicity against human RBC and MRC-5 cells. All
potencies are in M.
The specificity of AMP derived anticancer peptides for cancer
cell lines over non-transformed cell lines, or for specific cancer cell
lines, is based on different charge of the outside of the membrane
and the fluidity of the membrane as well as increased surface of the
membrane due to increased number of microvilli on cancer cells
[4]. Cancer cells are reported to have increased negatively charged
membranes due to a 3e9% excess of phosphatidylserine, increased
sialylation of membrane bound glycoproteins and glycolipids as
well as increased level of O-glycosylated mucins [32,33].
m
Entry
IC50
EC50
Selectivity index
RBC/Ramosd
Ramosa
RBCb
MRC-5c
MRC-5/Ramose
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
10a
10b
10c
11a
11b
11c
12a
12b
12c
10.6
7.1
4.1
14.4
11.0
4.3
63
78
79
136
>254
107
31
2.3
3.1
28
11.4
6.6
18
6.1
17.8
22
35
30
1.7
60
112
17.6
72
0.6
2.5
5.4
2.4
2.8
1.5
1.0
2.2
9.6
1.2
425
457
254
790
447
n.s.
n.s.
n.s.
>654
990
425
52
437
458
274
468
217
337
1116
409
349
526
303
6.6
64
104
The
b
2,2-amino acid derivative 5c was selected based on its
171
760
158
>634
552
90
8.2
12.7
49
structural simplicity, high potency and selectivity to obtain infor-
mation concerning its activity against different cancer cell lines,
and was screened against 59 cancer cell lines at the National Cancer
Institute (NCI), USA. The results revealed that 5c was active against
all 59 cancer cell lines tested, and displayed IC50 values between
>4.8
4.0
1.7
190
148
9.8
41
33
65
58
57
5.2
2.9
3.6
4.1
1.8
4.1
0.7
2.3
3.6
2.6
2.7
6.7
2.9
0.32
mM and 3.89 mM (Fig. 2). Compound 5c was exceptionally
potent against all the colon cancer cell lines COLO 205, HCC-2998,
HCT-116, HCT-15, HT29, KM12, and SW-620, the non-small cell
lung cancer cell line NCI-H460, the melanoma cell line LOX IMVI,
and the three leukemia cell lines HL-60(TB), K-562, and SR. The IC50
47
4.9
5.2
19
7.2
6.1
27
14.0
12.0
69
18.5
16.6
182
41
values against all these cell lines were below 1 mM.
In addition to the IC50 results, the NCI also provided an LC50
value, corresponding to the concentration where 50% of the starting
cells were killed (Figures for the LC50 results from NCI are shown in
57
19.6
22
the Supporting information). The LC50 results were below 8
all the cell lines, except for the leukemia cell lines, which displayed
LC50 values of 9.1 M for one cell line and above 10 M for the
mM for
n.s.: not soluble to required concentration in test media.
a
IC50 values against the Ramos cancer cell line.
EC50 values against human RBC.
EC50 values against the MRC-5 cell line.
b
m
m
c
remaining five cell lines. Despite the low IC50 values against the
colon cancer cell lines, the LC50 values were in the same range as
against the other cancer cell lines, resulting in a noticeable differ-
ence between the LC50 and the IC50 values for the colon cancers.
Furthermore, a TGI value (total growth inhibition) was provided
by NCI and corresponded to the concentration where there was no
detectable growth during the course of the screening (Figures of
TGI are shown in the Supporting information). The TGI values were
in average 1.3 times higher than the IC50 values, but still below
d
Selectivity index calculated as the EC50 value against human RBC divided by the
IC50 value against the Ramos cell line.
e
Selectivity index calculated as the EC50 value against normal MRC-5 cells
divided by the IC50 value against the Ramos cancer cell line.
whether they penetrate the membrane. In total, these properties
determine whether the derivatives kill cancer cells through
a membranolytic mode-of-action or interaction with intracellular
targets, as demonstrated for the antimicrobial and anticancer
peptide LfcinB [16]. However, extensive future experiments such as
calcein release studies from model liposomes are required to
establish the details of membrane interactions for individual
5 mM. Also for the TGI values, the leukemia cell lines and the colon
cancer cell lines displayed the largest differences when compared
to the IC50 values. The small differences between the IC50 and the
TGI and LC50 (Figures enclosed in the Supporting information) were
consistent with our previous data on bacteria, in which very small
differences were seen between the minimum inhibitory concen-
tration (MIC) and the minimum bactericidal concentration (MBC)
[21]. Larger AMPs are known to exert their antibacterial effects
through a killing mode of action, and the small differences
observed in this study between the IC50, the TGI, and the LC50
b
2,2-amino acid derivatives.
2.3. Hemolytic activity and toxicity against MRC-5 cell
The
2,2-amino acid derivatives were in general non-toxic to
b
human RBC and displayed EC50 values above 200 M, which was
well above their anticancer potencies. The only exception was 5a,
which previously has been shown to be surprisingly hemolytic with
m
values supported a cytotoxic mode-of-action for the
acid derivatives, and not a cytostatic mechanism [34].
b
2,2-amino
an EC50 value of 18
mM [22].
The MRC-5 cells were in general less susceptible to the b2,2
-
The COMPARE database supplied by the NCI revealed further-
more that 5c was on average almost 4 times more potent than
tamoxifen against 58 of the 59 cell lines. In addition, 5c was on
average 50 times more potent than the widely used antimetabolite
5-fluorouracil (5-FU). It should, however, be noted that the poten-
cies of 5-FU displayed more variation than for both 5c and
tamoxifen, probably due to a different mode of action for 5-FU.
amino acid derivatives compared to the Ramos cells. In general, the
most potent derivatives against the Ramos cancer cells were also the
most potent derivatives against the MRC-5 cells, but 5c, 9b, and 9c
were anyway 3.6e5.4 fold more potent against the Ramos cancer cell
line than the MRC-5 cells. Of note,10c showed the highest toxicity of
all derivatives against the non-malignant MRC-5 cell line, and was in
fact more toxic against MRC-5 cells than against Ramos cells.
In order to compare the toxicity and the potency of the
2.5. Conclusion
b
2,2-amino acid derivatives, we calculated a selectivity index (SI) by
dividing the IC50 value against the Ramos cancer cell line with the
EC50 values against RBCs and MRC-5 cells (Table 1). Among deriv-
Design of small amphipathic scaffolds is an extensively used
strategy for preparing small AMP-based antimicrobial peptidomi-
metics with improved pharmacokinetic properties, but the litera-
ture is limited with respect to applying the same strategy to design
small anticancer peptidomimetics [2,21,22,27,35e39]. We have,
atives with IC50 values below 8
mM, highest selectivity for the
Ramos cancer cell line compared to RBCs was 9b, whereas highest
selectivity compared to MRC-5 cells was 5c.

Fig. 2. Results from screening of 5c against 59 cancer cell lines at the NCI. Derivative 5c showed exceptional potency with IC50 values below 1 mM against the colon cancer cell lines,
the non-small cell lung cancer NCI-H460, the melanoma LOX IMVI cell line, and the three leukemia cell lines HL-60(TB), K-562, and SR.

28
T. Hansen et al. / European Journal of Medicinal Chemistry 58 (2012) 22e29
however, in the present study shown that small amphipathic b2,2
-
calculated as the ratio of compound treated sample to the Triton
X-100 treated sample.
amino acid derivatives fulfilling the pharmacophore model of small
cationic AMPs are highly potent against cancer cells. A recent
mode-of-action study has also shown that both a membranolytic
mechanism and interaction with intracellular targets are possible
3.2.2. Cell lines and cultures
Ramos human Burkitt’s lymphoma cells were grown in
suspension using RPMI 1640-medium supplemented with 10% fetal
bovine serum (FBS). The MRC-5 human embryonic lung fibroblasts
were cultured as monolayer with 10% FBS supplemented Minimal
Essential Medium (MEM). Both cell lines were maintained in
a humidified incubator at 37 ꢀC and 5% CO2. Both cell lines were
kindly provided by Prof. Øystein Rekdal.
based on the amphipathic nature of the b
2,2-amino acid derivatives
[29]. The results of these studies show that 5c is a promising
anticancer peptidomimetic with high anticancer potency, and high
selectivity for Ramos cancer cells compared to RBCs and MRC-5
cells. Screening of 5c against 59 different cancer cell lines at the
NCI confirmed the anticancer potency, and revealed that 5c
displays a broad range of activity against different cancer cell lines.
Thus, design of small amphipathic
b
2,2-amino acids and related
3.2.3. MTT assay
scaffolds is a promising strategy also for developing new anticancer
agents. An important future project in addition to further toxicity
A colorimetric cell viability assay under usage of the tetrazole 3-
(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT)
was used to evaluate the cytotoxic effects of the test compounds
[40]. Ramos cells were counted and 0.1 ml of a 4 ꢁ 105 cells/ml cell
suspension was seeded in 96-well plates using serum free RPMI-
1640 medium. The cell concentration of MRC-5 cells was adjusted
to 1 ꢁ 105 cells/ml and 0.1 ml seeded with MEM medium supple-
mented with 10% FBS. In RPMI-1640 dissolved compounds were
added immediately to the Ramos cells in a volume of 0.1 ml, while
the MRC-5 cells were first cultured overnight, the medium removed
and 0.1 ml of compounds added. A 1% solution of Triton X-100 in
RPMI-1640 medium and pure RPMI-1640 medium were used as
positive and negative controls. The cells were incubated at 37 ꢀC
and 5% CO2 in a humidified incubator for 4 h and subsequently the
MTT work solution added. Incubation continued for additional 2 h.
For Ramos cells 0.13 ml, for MRC-5 cells 0.7 ml incubation medium
was removed and 0.1 ml acidic isopropanol (0.04 N HCl) added.
After resuspending all wells, the plates were set on an orbital
shaker rotating at 400 rpm and further incubated at room
temperature until the formazan crystals were completely dissolved.
Absorbance was measured via a multiwell spectrophotometer at
590 nm. The cell survival rate was calculated as ratio of the back-
ground corrected absorbance values of compound treated cells and
the absorbance of the negative control (100% living cells). Following
the half inhibitory concentration (IC50) was determined.
studies, is to investigate if such small anticancer b
2,2-amino acid
derivatives are able to activate adaptive immunity in vivo against
established tumors, as reported for a LfcinB derivative [17].
3. Experimental section
3.1. Chemistry in general
Chemicals and solvents were purchased from SigmaeAldrich
and used without further purification. 1H and 13C NMR spectra
were recorded on a 400 MHz Varian spectrometer. Chemical shifts
are expressed in ppm relative to CHCl3 (1H 7.26 ppm, 13C 77.0 ppm)
or methanol (1H 3.31 ppm, 13C 39.0 ppm). The values are given in
d
scale. Mass spectra were obtained on a Micromass Quattro LC
(Micromass, Manchester, UK). High-resolution mass spectra were
obtained on a Waters Micromass LCT Premier mass spectrometer
(Micromass, Manchester, UK). Preparative RP-HPLC was carried out
on
a Waters 616 system equipped with a XBridge, 5 mm,
19 ꢁ 250 mm C18-column, and eluted with acetonitrile and water,
both containing 0.1% TFA. Analytical HPLC was carried out on
a Waters 2695 HPLC equipped with a Sunfire, 4.6 ꢁ 250 mm C18
-
column and analyzed at wavelengths 214 and 254 nm with a PDA
detector spanning from wavelengths 210 nme310 nm. All
compounds were prepared by using parallel reaction carousels
from Radleys. All compounds undergoing biological evaluation
possessed purity above 95% as determined by analytical RP-HPLC-
PDA. For synthesis of compounds 1aec, 2aec, 3aec, 5aec, 8aec,
10aec, 11aec and 12aec please see our previous publications in
Refs. [21,22]. Please see the Supporting information for details
regarding synthesis and in vitro testing of the new compounds 6ae
c, 7aec and 9aec described in the paper.
Acknowledgments
We thank Prof. Øystein Rekdal at the Tumor Biology Group,
University of Tromsø, for providing the Ramos and MRC-5 cell lines
and NCI for the extended screening of 5c. We also thank Dr. Espen
Hansen, (Marbio, University of Tromsø) for help with the HRMS
analysis.
3.2. In vitro testing
Appendix A. Supplementary data
3.2.1. Hemolytic activity
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.09.048.
Hemolytic activity was measured as previously described in Ref.
[21]. Briefly, heparinized human blood was centrifuged and washed
three times with 37 ꢀC pre-warmed phosphate buffered saline
(PBS) in order to remove the plasma fraction. The red blood cells
(RBCs) were diluted to 10% the hematocrit value in PBS and the
compounds under investigation were dissolved in the same buffer.
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