Antimicrobial activity of small β-peptidomimetics based on the pharmacophore model of short cationic antimicrobial peptides

Article abstract of DOI:10.1021/jm901052r

We have synthesized a series of small β-peptidomimetics (M_w <650) that were based on the minimal pharmacophore model for anti-Staphylococcal activity of short cationic antimicrobial peptides. All β-peptidomimetics had a net charge of +2 and formed an amphipathic scaffold consisting of an achiral lipophilic β^2,2-amino acid coupled to a C-terminal L-arginine amide residue. By varying the lipophilic side-chains of the β^2,2-amino acids, we obtained a series of highly potent β-peptidomimetics with high enzymatic stability against α-chymotrypsin and a general low toxicity against human erythrocytes. The most potent β-peptidomimetics displayed minimal inhibitory concentrations of 2.1-7.2 μM against Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant Staphylococcus epidermidis (MRSE), and Escherichia coli. Small amphipathic β-peptidomimetics may be a promising class of antimicrobial agents by means of having a similar range of potency and selectivity as larger cationic antimicrobial peptides in addition to improved enzymatic stability and lower costs of production.

Full text of DOI:10.1021/jm901052r

J. Med. Chem. 2010, 53, 595–606 595
DOI: 10.1021/jm901052r
Antimicrobial Activity of Small β-Peptidomimetics Based on the Pharmacophore Model of Short
Cationic Antimicrobial Peptides
Terkel Hansen, Tore Alst, Martina Havelkova, and Morten B. Strøm*
Department of Pharmacy, Faculty of Health Sciences, University of Tromsø, N-9037 Tromsø, Norway
Received July 16, 2009
We have synthesized a series of small β-peptidomimetics (Mw <650) that were based on the minimal
pharmacophore model for anti-Staphylococcal activity of short cationic antimicrobial peptides. All
β-peptidomimetics had a net charge of þ2 and formed an amphipathic scaffold consisting of an achiral
lipophilic β2,2-amino acid coupled to a C-terminal L-arginine amide residue. By varying the lipophilic
side-chains of the β2,2-amino acids, we obtained a series of highly potent β-peptidomimetics with high
enzymatic stability against R-chymotrypsin and a general low toxicity against human erythrocytes. The
most potent β-peptidomimetics displayed minimal inhibitory concentrations of 2.1-7.2 μM against
Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), methicillin resistant
Staphylococcus epidermidis (MRSE), and Escherichia coli. Small amphipathic β-peptidomimetics may
be a promising class of antimicrobial agents by means of having a similar range of potency and selectivity
as larger cationic antimicrobial peptides in addition to improved enzymatic stability and lower costs of
production.
Introduction
nonreceptor specific manner.14,15 The detailed mechanism of
membrane disruption by AMP’s is still not fully understood,
and various models have been proposed to explain the
observed effects such as the carpet, barrel-stave, and micelles-
aggregate models (see Giuliani et al. for an excellent review).14
However, the use of peptides as drugs is challenged by
deficiency in patient-friendly delivery technologies, low bio-
availability, low metabolic and proteolytic stability, high costs
of manufacture, and potential danger of immunological
responses.16 Currently there are only two peptide-based anti-
microbial drugs on the market, polymyxin B, indicated for
skin infections by Gram-negative bacteria and Daptomycin,
indicated for skin infections by Gram-positive bacteria.14
However, there is hope for the future, and three AMP’s are
currently in phase III clinical trials and yet another 12
compounds from 10 different companiesare inthe earlystages
of clinical trials.14
One method to overcome the obstacles of transforming
natural AMP’s into peptide-drugs has been to prepare shorter
derivatives with, e.g., a high content of basic and aromatic
amino acids. The minimal pharmacophore model of short
AMP’s ranging in size from two to six amino acid residues has
been established by the research group of Svendsen.17 The
pharmacophore model for such short AMP’s can be described
relatively simply in which activity against the Gram-negative
bacterium Escherichia coli (E. coli) necessitates an amphi-
pathic peptide with three bulky/lipophilic groups and two
positively charged groups.17 For activity against the Gram-
positive bacteria Staphylococcus aureus (S. aureus), MRSA
and MRSE, the pharmacophore is even simpler by only
necessitating an amphipathic peptide with two bulky/lipophi-
lic groups and two positively charged groups.17 Further
studies of the pharmacophore model for anti-staphylococcal
Infections caused by multiresistant bacteria have become a
major concern to society over the past 20-25 years.1,2 This is
especially a problem in hospitals where infections caused by
methicillin resistant Staphylococcus aureus (MRSAa), vanco-
mycin resistant Staphylococcus aureus (VRSA), and methi-
cillin resistant Staphylococcus epidermidis (MRSE) can result
in severe injury, prolonged hospitalization, and death, espe-
cially among immune compromised patients.3,4 Vancomycin
was once a drug of last resort, but reports from hospitals
around the world shows that infrequent use of vancomycin is
no longer the case.2,5,6 At the same time, there has been a
drought among the largest pharmaceutical companies for
developing novel classes of antimicrobial compounds, basi-
cally stating it to be a high-risk investment due to huge costs of
development and relatively limited duration of patient treat-
ment as opposed to treatment of chronic diseases.7-9 How-
ever, the need for novel antimicrobial agents is urgent, and in
the U.S., deaths caused by hospital acquired infections are
currently superseding HIV-related mortality.10
A promising class of antimicrobial agents is cationic anti-
microbial peptides (AMP’s), also known as host defense
peptides, which have been heavily investigated over the past
20 years.11-13 AMP’s have a unique mode of action by
targeting the inner and/or outer membranes of bacteria in a
*To whom correspondence should be addressed. Phone: þ47 77 64 40
85. Fax: þ47 77 64 61 51. E-mail: morten.strom@uit.no.
a Abbreviations: AMP, cationic antimicrobial peptide; DIPEA, di-
isopropyethylamine; MBC, minimal bactericidal concentration;
MRSA, methicillin resistant Staphylococcus aureus; MRSE, methicillin
resistant Staphylococcus epidermidis; Ra/Ni, Raney nickel catalyst;
RBC, red blood cells; TEA, triethylamine; TFFH, fluoro-N,N,N0,N0-
tetramethylformamidinium hexafluorophosphate; TIS, triisopropylsi-
lane.
r
2009 American Chemical Society
Published on Web 12/08/2009
pubs.acs.org/jmc

596 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Hansen et al.
Scheme 1. Overview of Synthesis of the β-Peptidomimetics
4a-na
a (a) NaOMe, R-Br, performed twice, 78 °C s: MeOH. (b) Ra/Ni, H2
(g), 45 °C, 5 days, s: MeOH. (c) TEA, pH 8, Boc2O, rt, 18 h, s: H2O:
dioxane (1:5). (d) LiOH, 18 h, 100 °C, s: H2O:dioxane (1:3). (e) DIPEA,
TFFH, rt, s: DMF. (f) H-Arg-NH2 ꢀ 2 HCl, DIPEA, rt, 7 days, s:
DMF. (g) TFA: TIS: H2O (95:2.5:2.5), rt 2 h, s: DCM.
shown in Scheme 1 were isolated (side-chain structures are
shown in Figure 1). Synthesis of the β2,2-amino acid methyl
esters (steps a and b) were based on the studies by Cronin
et al.,19 and the chosen strategy made it possible to use
parallel-synthesis of all compounds on a 6- or 12-position
Radleys reaction carousel. The critical step in our synthesis
was the hydrolysis of the most lipophilic β2,2-amino acid
methyl esters due to their low solubility. The problem was
partly solved by individually adjusting the ratio between
water and 1,4-dioxane for each compound, as described in
the Experimental Section. The total yields for synthesis of the
crude Boc-β2,2-amino acids (3a-n) ranged therefore from 30
to 71%, with the exception of compound 3k, which gave a
total yield of 15%, and compound 3b, which gave a total
yield of only 5%. To improve the overall yields of the Boc-
β2,2-amino acids (3a-n), we also tried benzyl and allyl esters
that could be removed together with the reduction of the
nitrile. However, we experienced that the following Boc
protection did not work satisfactorily. The reason for this
was not investigated in detail, but we suspected that it may
have been caused by complexation between nickel ions (from
the Ra/Ni catalyst) with the free amino- and carboxylic acid
groups of the β2,2-amino acids.20,21
Because of sterical hindrance of the carboxyl group of the
Boc-protected β2,2-amino acids, we performed coupling of
the C-terminal arginine amide residue through an acid
fluoride intermediate according to Carpino and El-Faham.22
However, we found it mandatory to increase the reaction
time for preactivation with TFFH from 15 min to 2 hours
and to increase the coupling time from 1 h to up to 7 days for
the most sterically hindered derivatives, as determined by
studying the progress of the reaction by MS-analysis. After
acidic cleavage of the Boc-protecting group, the crude
β-peptidomimetic was purified by RP-HPLC to a final purity
of >95% as determined by analysis with an analytical RP-
HPLC C18-column and UV detection at 214 and 254 nm.
Antimicrobial Activity. The results from screening of anti-
microbial activity showed that the β-peptidomimetics were
in general more potent against the Gram-positive bacteria
than against the Gram-negative bacterium E. coli (Figure 1
and Table 1). A closer examination of the results revealed
that the overall most potent β-peptidomimetic was com-
pound 4n, which contained two super-bulky 3,5-di-tert-butyl
Figure 1. β-Peptidomimetics prepared for investigations of anti-
microbial activity. All compounds were designed to fulfill the
pharmacophore model of short AMP’s with anti-staphylococcal
activity and were based on a scaffold consisting of a lipophilic
β2,2-amino acid coupled to a C-terminal L-arginine amide residue.
activity have shown that the bulky groups should be aromatic
and have a size larger than a benzyl group.18
The purpose of the current project was to address some of
the general problems associated with peptide drugs by design-
ing novel antimicrobial compounds based on the pharmaco-
phore modelfor anti-staphylococcal activity. The result of our
investigations was a novel class of highly potent antimicrobial
β-peptidomimetics based on the scaffold of an achiral lipo-
philic 3-amino-2,2-disubstituted propionic acid (β2,2-amino
acid) coupled to a C-terminal L-arginine amide residue
(Figure 1). The overall size of the resulting β-peptidomimetics
resembled the size of a dipeptide while having the side-chain
functionalities of a tripeptide due to the β2,2-amino acid
derivative. A wide range of lipophilic substituents of the
β2,2-amino acid derivative was explored as shown in Figure 1.
The results demonstrated that the scaffold was highly efficient
for preparing novel antimicrobial compounds based on the
pharmacophore model of short AMP’s and with particularly
high potency against the Gram-positive bacteria S. aureus,
MRSA, and MRSE.
Results
Synthesis. The β-peptidomimetics were prepared in a
seven-step synthesis in which only the three intermediates

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 597
Table 1. Minimal Inhibitory Concentration (MIC) against S. aureus,
MRSA, MRSE, and E. coli and EC20 Values against Human RBC for
Antimicrobial β-Peptidomimetics Prepared in the Study
β-peptidomimetics were active against the Gram-negative
bacterium E. coli within the concentration range tested.
When comparing the para-substituted benzylic β-peptido-
mimetics, compounds 4h, 4i, 4j, and 4k, the potency of the
β-peptidomimetics increased by increasing the size and
lipophilicity of the para-substituents. As described above,
compound 4k with two para-tert-butyl benzylic side-chains
was the overall second most potent β-peptidomimetic
against both the Gram-positive and Gram-negative bacteria.
The less bulky compound 4j with two para-isopropyl
benzylic side-chains showed similar potency as compound
4k against the Gram-positive bacteria but was much less
potent against E. coli than compound 4k.
MICa (μM)
EC20b (μM) therapeutic
entry S. aureusc MRSAd MRSEe E. colif
RBCg
indexh MRSA
4a
4b
4c
-
150
-
150
-
75
-
-
-
-
-
-
10h
200h
7.2
7.2
7.2
144
4d
4e
4f
-
147
49
-
147
49
-
74
35
-
-
nt
-
-
10h
29h
52
-
266
-
259
4g
6.6
5.0
6.6
The least potent β-peptidomimetic among the para-sub-
stituted benzylic derivatives was compound 4h, which con-
tained two para-methyl benzylic side-chains in the
β2,2-amino acid derivative. It was noteworthy that the po-
tency of compound 4h was comparable to the potency of
compound 4e, in which the total lengths of the side-chains
were approximately the same but differed structurally
(Figure 1). A small difference in potency against MRSA
may have been influenced by this structural difference
because compound 4h was a little more potent against
MRSA, but both of these β-peptidomimetics were inactive
against E. coli within the concentration range tested.
A 6-23-fold increase in potency against the Gram-posi-
tive strains was obtained by replacing the para-methyl
benzylic side-chains in compound 4h with two para-tri-
fluoromethyl benzylic side-chains. The resulting compound
4i displayed also measurable activity against E. coli.
4h
4i
4j
4k
147
12.7
74
3.2
74
-
254
136
46
-
756
254
578
20h
236
37
12.7
6.8
5.0
10.2
6.5
6.8
3.3
175
4l
4m
4n
60
259
2.9
49
259
2.1
35
129
10.1
-
-
-
-
29h
5h
5.7
51
24
a Highest concentration tested was 200 μg/mL. b Highest concentra-
tion tested was 1000 μg/mL. c Staphylococcus aureus (ATCC 25923).
d Methicillin resistant Staphylococcus aureus (ATCC 33591). e Methi-
cillin resistant Staphylococcus epidermidis (ATCC 27626). f Escherichia
coli (ATCC 25922). g Human red blood cells. h Therapeutic index was
calculated as the hemolytic activities (EC20) divided by the MIC values
against MRSA. For nonhemolytic compounds, the EC20 value was set to
an arbitrary value of 1000 μg/mL in order to calculate a therapeutic
index. The notation “-” denotes no detectable activity (MIC or EC20
within the concentration range tested. nt: not tested.
)
The final three β-peptidomimetics shown in Figure 1
contained two 3,5-disubstituted benzylic side-chains in the
β2,2-amino acid derivatives, and as described above, the
largest β-peptidomimetic compound 4n was also the overall
most potent β-peptidomimetic prepared in the study. Com-
pound 4l, containing two 3,5-dimethyl benzylic side-chains,
had the same molecular mass as the elongated aromatic
compound 4f and showed similar potency against the
Gram-positive bacteria. However, neither compounds 4l
nor 4f showed antimicrobial activity against E. coli.
Despite its large size and high molecular mass, compound
4m containing two 3,5-dimethoxy benzylic side-chains
showed very poor Gram-positive activity and no antimicro-
bial activity against E. coli within the concentration range
tested. Seemingly, the polar methoxy substituents in the
benzylic side-chains of the β2,2-amino acid constituting the
lipophilic part of the scaffold had a negative influence on
antimicrobial potency.
Hemolytic Activity. Hemolytic activity against human red
blood cells (RBC) was used as a measurement of toxicity, but
due to a general low hemolytic activity of the majority of the
β-peptidomimetics, the threshold of detection was changed
from the standard 50% hemolytic activity (EC50) to mea-
surement of 20% hemolytic activity (EC20). Furthermore, a
therapeutic index was calculated based on the EC20 results
against human RBC and the antimicrobial activity against
MRSA (Table 1).
The results showed that only compounds 4g, 4i, 4j, 4k, and
4n displayed measurable EC20 values, whereas all the other
β-peptidomimetics prepared showed no measurable hemo-
lytic activity within the concentration range tested, i.e., up to
1000 μg/mL. A comparison of the hemolytic profile of these
five β-peptidomimetics is shown in Figure 2. Highest hemo-
lytic activity was displayed by the super-bulky compound 4n,
benzylic side-chains in the β2,2-amino acid derivative. It is
noteworthy that this was the only β-peptidomimetic that
showed the same high potency against both Gram-positive
and Gram-negative bacteria. The somewhat less bulky
β-peptidomimetic compound 4k, which contained two
para-tert-butyl benzylic side-chains, showed the second high-
est antimicrobial potency against all Gram-positive bacteria
and the Gram-negative bacterium E. coli. The naphthalene
derivative compound 4g was also among the most potent
anti-staphylococcal β-peptidomimetics prepared but much
less potent against E. coli.
A somewhat surprising high antimicrobial potency against
the Gram-positive strains was displayed by compound 4c,
although the activity against E. coli was poor. In contrast to
the β-peptidomimetics described above, compound 4c was
not aromatic but belonged to the group of aliphatic
β-peptidomimetics by containing two cyclohexyl-ethyl side-
chains in the lipophilic part of the molecule. The related
aliphatic compound 4b showed, however, poor anti-staphylo-
coccal activity and no antimicrobial activity was detec-
ted against E. coli. The least bulky derivative within the
group of aliphatic β-peptidomimetics, compound 4a, was
inactive against all test bacteria within the concentration range
tested.
A similar increase in potency as observed for compounds
4b and 4c by increasing the length of the lipophilic side-
chains was observed for the aromatic compounds 4e and 4f.
Although compound 4d was inactive against all test strains,
the longer β-peptidomimetic compound 4e displayed anti-
staphylococcal activity, with highest potency obtained
against MRSE. Further side-chain elongation resulted in a
more than 2-fold improvement in antimicrobial potency as
observed for compound 4f. However, none of these

598 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Hansen et al.
used as bioisosteric replacements to improve the pharmaco-
kinetic properties of biological active peptides such as AMP’s.
In the current study, we chose to investigate a scaffold that
would take advantage of the profound enzymatic stability
reported for β-peptides and at the same time fulfill the
pharmacophore model of short AMP’s with anti-staphylo-
coccal activity.17 The scaffold was based on an achiral lipo-
philic β2,2-amino acid coupled to a C-terminal L-arginine
amide residue, which ensured that all the β-peptidomimetics
consisted of two lipophilic groups and had a net charge of þ2.
The resulting β-peptidomimetics were thereby bioisosteres of
dipeptides but having the side-chain functionalities of tripep-
tides due to the β2,2-amino acid derivatives.
Figure 2. Hemolytic activity of the five β-peptidomimetics that
displayed hemolytic activity below 1000 μg/mL. No hemolytic
activity at all was detected for any of the other β-peptidomimetics.
All β-peptidomimetics were tested for hemolytic activity up to a
concentration of 1000 μg/mL, except for compounds 4k (tested up
to 500 μg/mL) and 4n (tested up to 71.5 μg/mL) due to limited
solubility in the test media.
The selection of arginine and not lysine as cationic residue
was based on the presumption that the guanidine group of
arginine would interact more strongly with the negatively
charged phospholipids of the bacterial cell membrane by
forming both electrostatic and hydrogen-bonding interac-
tions.31 Furthermore, the choice of a C-terminal arginine
residue and not an N-terminal arginine residue was to further
protect the β-peptidomimetics against proteolytic degrada-
tion by, e.g., trypsin. Trypsin has an anionic specificity-pocket
favoring binding to cationic amino acids and hydrolysis of the
amide bond between the cationic residue and the next amino
acid in the sequence.32 In this way, the β-peptidomimetics
prepared would form poor substrates for trypsin. However,
the N-terminal lipophilic β2,2-amino acids could render the
β-peptidomimetics susceptible to degradation by other pro-
teases, e.g., chymotrypsin. Chymotrypsin has a lipophilic
specificity pocket and normally hydrolyzes amide bonds
between lipophilic/aromatic amino acids such as tyrosine,
phenylalanine, tryptophan, or leucine, and the following
amino acid in the sequence.33 Thus, the resemblance of some
of the β2,2-peptidomimetics to naturally occurring lipophilic/
aromatic amino acids could render them substrates for chymo-
trypsin. To investigate this, we selected six different
β-peptidomimetics (4c, 4g, 4h, 4i, 4k, and 4m) and treated
them with R-chymotrypsin. The results revealed that no
proteolytic digestion could be detected for any of the com-
pounds within the experimental time frame of 48 h (Figure 3).
The results were in agreement with other studies on pure
β-peptides, and in our case the protection against proteolytic
degradation could be explained by the sterical hindrance
offered by the disubstituted R-carbon of the β2,2-amino acids,
the increased distance between the N-terminal amino group,
and the carboxyl group of the β2,2-amino acids, or that the
β-peptidomimetics simply were too small to bind to the active
site of the enzyme.
On the basis of the results of the β-peptidomimetics, the
close spatial localization of the two lipophilic side-chains of
the β2,2-amino acid residues seemed to be an efficient motif for
preparing highly potent antimicrobial β-peptidomimetics.
Wessolowski et al. have recently shown that three adjacent
aromatic residues in short arginine- and tryptophan-rich
AMP’s is an efficient structural motif for ensuring high
antimicrobial potency.34 The scaffold we have developed
enabled an even more closely localization of the lipophilic
groups by bringing together two lipophilic side-chains on the
same R-carbon in a single β2,2-amino acid residue.
An amphipathic structure is often essential for antimicrobial
activity, and most natural AMP’s obtain this by forming larger
secondary structures such as R-helixes or β-sheets.15,35-37
Irrespective of conformation, the β-peptidomimetics pre-
pared in the present study would be able to form strong
Figure 3. Degradation of six selected β-peptidomimetics by
R-chymotrypsin. The ratio was calculated as remaining substance
in samples with enzyme divided by samples without enzyme; 100%
equals no enzymatic degradation. Only samples collected at 0, 60,
120 min and 24 and 48 h are shown.
but the therapeutic index revealed nevertheless 24-fold selec-
tivity for MRSA compared to human RBC.
To calculate a therapeutic index for all the β-peptido-
mimetics, the EC20 value of the nonhemolytic compounds
was set to an arbitrary EC20 value of 1000 μg/mL (Table 1).
When comparing the β-peptidomimetics 4c, 4g, 4i, 4j, 4k, and
4n that all showed MIC values below 10 μM against MRSA,
the highest therapeutic index was displayed by compound 4i
containing two para-trifluoromethyl benzylic side-chains
followed by compound 4c, which contained two cyclo-
hexyl-ethyl side-chains, and compound 4k finishing third
with two para-tert-butyl benzylic side-chains in the β-amino
acid derivative.
Stability. A selection of six β-peptidomimetics (4c, 4g, 4h,
4i, 4k, and 4m) was investigated for proteolytic degradation
by R-chymotrypsin (Figure 3). The results from evaluation of
proteolytic stability against R-chymotrypsin revealed that no
degradation could be detected for any of the compounds
over a period of 48 h. We also found that all test compounds
were chemically stable in aqueous solutions at pH 7.4 for at
least 48 h.
Discussion
The research field on antimicrobial β-peptides or β-pepti-
domimetics is relatively new, and only a limited number of
research groups have reported design of novel antimicrobial
β-peptides.23-27 Tests in vivo and in vitro have shown that
β-peptides are highly stable against proteolytic degradation
by human enzymes such as trypsin, chymotrypsin, and
elastase.28-30 Incorporation of β-amino acids can thereby be

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 599
bulky isopropyl group on the benzylic side-chains of com-
pound 4j ensured sufficient bulkiness for a high potency
against the Gram-positive bacteria, although the potency
against E. coli was reduced compared to compound 4k. The
importance of side-chain structure was also evident for the
naphthalene derivative compound 4g, which was the third
most potent β-peptidomimetic against the Gram-positive
bacteria but displayed relatively low potency against the
Gram-negative bacterium E. coli.
The perhaps largest increase in potency by a single side-
chain modification was the replacement of the para-methyl
benzylic side-chains of compound 4h with the para-trifluoro-
methyl benzylic side-chains, resulting in compound 4i. The
effect of the trifluoromethyl substitution on antimicrobial
potency was pronounced and resulted in a 23-fold improve-
ment in antimicrobial potency against MRSA, more than
11-fold improvement against S. aureus, a 6-fold improvement
against MRSE, and detectable activity against E. coli. Sub-
stitutionofbenzylicor aromatichydrogenatomswithfluorine
is often used during drug optimization to increase lipophilicity
or modulate basicity, or perhaps more importantly to impede
metabolism by human CYP450 enzymes.38-40 Oxidation by
CYP450 enzymes often results in formation of inactive meta-
bolites and increased renal excretion of active metabolites due
to increased polarity. In the present case, the trifluoromethyl
group of compound 4i may result in increased metabolic
stability compared to compound 4h by preventing CYP450
oxidation of the side-chains to phenylmethanol, benzal-
dehyde, or benzoic acid derivatives.41,42 Furthermore, the
latter oxidation to a carboxylic acid group in the bulky part
of the β-peptidomimetics would not only disrupt the amphi-
pathicity of the molecule but also reduce the overall net
positive charge and result in metabolites that no longer
fulfilled the pharmacophore model for anti-staphylococcal
activity. Thus, compound 4i is a promising compound for
further investigations by being highly potent against bacteria
as a result of high lipophilicity and by possessing improved
pharmacokinetic properties with respect to likely increased
metabolic stability and bioavailability in vivo.43,44
Further structural alterations, which resulted in increased
antimicrobial potency, involved elongation of the side-chains
of the β2,2-amino acid derivatives, as observed for the increas-
ingly elongated and more potent aromatic β-peptidomimetics
4e and 4f. It was rather surprising that such structural altera-
tions also applied to the aliphatic β-peptidomimetics com-
pounds 4b and 4c and that the effect of the side-chain
elongation of the aliphatic β-peptidomimetics was even more
evident than for the aromatic β-peptidomimetics. The alipha-
tic compound 4c had two side-chains consisting of elongated
ethyl-cyclohexyl groups and was the fourth to fifth most
potent β-peptidomimetic against both the Gram-positive
and Gram-negative bacteria. Thus, compound 4c was 10- to
20-fold more potent against the Gram-positive bacteria than
its aromatic counterpart compound 4e. A similar effect was
observed against S. aureus and MRSA for the aliphatic
compound 4b, which was more potent than its aromatic
counterpart compound 4d. Previous studies on AMP’s have
mainly focused on aromatic side-chains due to their prefer-
ence for localizing in the interface region of the phospholipid
bilayer of the bacterial cell membrane.15,35,45 However, the
results of the present study showed that aliphatic side-chains
containing cyclohexyl groups can give even more potent
derivatives although possibly not having a preference for the
membrane interface region. With regard to interaction with
Figure 4. Relationship between antimicrobial potency (1/MIC)
against S. aureus and the retention time (Rt) of the compounds on
an analytical RP-HPLC C18 column as a measurement of overall
lipophilicity.
amphipathic structures upon interaction with a bacterial cell
membrane by having a cationic N-terminal amino group and
a C-terminal arginine amide residue flanking the lipophilic
core of the scaffold. The only β-peptidomimetic that deviated
from the requirement of a strong amphipathicity was com-
pound 4m, which contained two relatively polar 3,5-di-
methoxy benzylic side-chains. As a consequence, compound
4m displayed much lower antimicrobial potency compared
to other structurally related β-peptidomimetics prepared,
such as compound 4l containing two 3,5-dimethyl benzylic
side-chains.
The results demonstrated a strong correlation between
antimicrobial potency and overall lipophilicity of the β-peptido-
mimetics prepared. This can be illustrated by a comparison
between the potency of the β-peptidomimetics against
S. aureus and their retention time (Rt) on an analytical RP-
HPLC C18 column, which demonstrated the affinity of the
β-peptidomimetics for the hydrophobic stationary phase of
the column (Figure 4). The synthesized β-peptidomimetics
could roughly be grouped into three classes based on their
potency and Rt. The least potent β-peptidomimetics com-
pounds 4b, 4e, 4f, 4h, 4l, and 4m had Rt between 15.5 and 19.3
min, the second most potent β-peptidomimetics compounds
4c, 4g, 4i, 4j, and 4k had Rt between 19.9 and 24.2 min,
whereas the most potent β-peptidomimetic compound 4n
formed a class in solitude by being most lipophilic and having
a Rt of 32.7 min. However, the correlation between antimi-
crobial potency and overall lipophilicity was not straightfor-
ward because the relatively long Rt of compound 4c should
indicate a much higher antimicrobial potency against
S. aureus. Similarly, one would expect a much lower anti-
microbial potency for the naphthalene derivative, compound
4g, based on its relatively short Rt.
In addition to the importance of overall lipophilicity,
certain structural alterations of the achiral lipophilic
β2,2-amino acid residues had an important impact on anti-
microbial potency. Clearly, the superbulkiness of the tert-
butyl-groups on the benzylic side-chains of compounds 4k
and 4n was important to the high potency and the broad range
of activity against both Gram-positive and Gram-negative
bacteria that these compounds showed. The somewhat less

600 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Hansen et al.
the bacterial cell membrane, the aliphatic compounds will
probably be more prone to interact with the aliphatic acyl-
chain region in contrast to the membrane interface region and
thereby cause a more efficient distortion of the intramolecular
organization of the phospholipids in the cell membrane. The
highly potent β-peptidomimetics containing tert-butyl groups
(4k and 4n) may combine the effects of the aliphatic com-
pounds and the less bulky aromatic compounds by being
partly aromatic and partly aliphatic.
Gram-negative bacteria E. coli, which is consistent with what
has been reported for many short AMP’s.17 We experienced
very small differences in MIC and MBC values, i.e., one titer-
step or less, which is not uncommon to short AMP’s and may
indicate that killing is the main bacteriostatic mode of action
(MBC results are shown in the Supporting Information).
Another important observation shared by short AMP’s was
that compounds of similar molecular mass were almost
equally potent.18 This was observed when comparing the
potencies of compounds 4e and 4h (Mw’s of 453.3) and
compounds 4f and 4l (Mw’s of 481.3), which were constitu-
tional isomers and displayed similar MIC values. A similar
mass-balance relationship has been observed for short
AMP’s, showing that there are small differences in potency
with respect to constitutional isomers as long as the require-
ments of a net positive charge and ability to form amphipathic
structures are fulfilled.48
We used hemolytic activity as a measurement of toxicity
against mammalian cells, but due to a general low hemolytic
activity, we chose to measure 20% hemolysis (EC20) in place
of the standard measurement of 50% hemolysis (EC50). Only
compounds 4g, 4i, 4j, 4k, and 4n displayed measurable
hemolytic activity, whereas no hemolytic activity at all could
be detected for the other β-peptidomimetics prepared up to
1000 μg/mL. Highest hemolytic activity was displayed by
compound 4n that, as described above, was the overall most
potent β-peptidomimetic against all bacterial strains and also
the most lipophilic compound prepared. It was noteworthy
that compounds 4j and 4k, which displayed comparable
antimicrobial activities against the Gram-positive bacteria,
showed dissimilar hemolytic activity. Haug et al.46 have
recently reported a series of synthetic di- and tripeptides
displaying MIC values of 3.2-28 μM against MRSA and
shown that the hemolytic activity (EC50) varies from 97 μM to
above the detection limit of 1000 μg/mL, i.e., nonhemolytic.
Thus, on the basis of the results from Haug et al. and the
present study, minor structural modifications of the lipophilic
groups of small AMP-based molecules can have a large effect
on hemolytic activity, whereas antimicrobial activity is much
less affected.
The EC20 measurements were also used to calculate a
therapeutic index with respect to antimicrobial activity
against MRSA and in which case the hemolytic activity of
the nonhemolytic compounds was set to an arbitrary EC20
value of 1000 μg/mL. On the basis of a total evaluation of
antimicrobial activity against MRSA, metabolic stability, and
the therapeutic index, compound 4i containing two para-
trifluoromethyl benzylic side-chains, and with a therapeutic
index of 236, appeared as the most promising β-peptidomi-
metic. In the case of antimicrobial activity against MRSE,
compound 4k emerged as the most promising candidate, with
a therapeutic index of 116 (calculation not shown). With
respect to the Gram-negative bacterium E. coli, a therapeutic
index of 12.5 was displayed by compound 4k while the much
more potent β-peptidomimetic 4n had a therapeutic index of
8.9 (calculations not shown). Thus, depending on the patho-
genic bacteria in question, different β-peptidomimetics should
be used in further studies based on a total evaluation of
antimicrobial potency, therapeutic index, and pharmaco-
kinetic properties.
Tew et al.49 have recently reported a study on AMP-mimics
basedonpolymeric phenylacetylenes and have shownthatthe
smallestphenyl acetylene derivative containingthree aromatic
rings and having a net charge of þ2 displays the highest
antimicrobial potency. It is noteworthy that this compound
was slightly more potent against E. coli than against S. aureus,
although polymeric derivatives of the same series showed the
opposite effect. The present study and the study by Tew et al.
demonstrate that different types of amphipathic scaffolds can
be used to design highly potent mimics of short AMP’s but
with improved pharmacokinetic properties.
Conclusion
We have prepared a series of highly potent β-peptidomi-
metics that were based on the pharmacophore model of short
AMP’s with anti-staphylococcal activity.17 The β-peptidomi-
metics consisted of a lipophilic β2,2-amino acid coupled to a
C-terminal L-arginine amide residue, ensuring a net positive
charge of þ2. The β-peptidomimetics were amphipathic as a
consequence of the close association of the lipophilic side-
chains on the same R-carbon of the β2,2-amino acid residue
and by having the lipophilic side-chains positioned between
two cationic groups. Amphipathicity is a crucial feature for
efficient membrane interaction by naturally occurring AMP’s
and also for selectivity for bacterial cells compared to human
cells.14,35 We observed a strong correlation between anti-
microbial potency and overall lipophilicity of the β-peptido-
mimetics, but structural properties of the β2,2-amino acid side-
chains were also highly decisive for the antimicrobial potency.
Side-chainmodifications thatgavethe mostpotent derivatives
included tert-butyl benzyl groups (4k and 4n), naphthalene
groups (4g), aliphatic cyclohexyl groups (4c), and trifluoro-
methyl benzyl groups (4i).
On the basis of an evaluation of the therapeutic index for
β-peptidomimetics displaying MIC values below 10 μM
against the Gram-positive test-bacteria, compound 4i con-
taining two para-trifluoromethyl benzylic side-chains
emerged as the most promising β-peptidomimetic against
MRSA infections, with a therapeutic index of 236. However,
compound 4k containing two para-tert-butyl benzylic side-
chains appeared as the most promising candidate against
MRSE infections, with a therapeutic index of 116. Compound
4n was the most potent β-peptidomimetic against the Gram-
negative bacterium E. coli and had a therapeutic index of 8.9
compared to human RBC. Although compound 4n had a
lower therapeutic index against E. coli than compounds 4i and
Although determination of the precise mechanism of action
of the β-peptidomimetics was not a subject of our study, the
β-peptidomimetics reported herein share many similarities
with observations from short AMP’s and may therefore
indicate a resembling mechanism of action. All β-peptidomi-
metics prepared in the present study fulfilled the pharmaco-
phore model of short AMP’s with anti-staphylococcal activity
and the general requirements of larger AMP’s by having a net
charge of þ2 and an amphipathic structure.34,47 As shown in
Table 1, all β-peptidomimetics prepared except for com-
pound 4n were more potent against the Gram-positive
bacteria S. aureus, MRSA, and MRSE than against the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 601
4k against the Gram-positive bacteria, compound 4n is a
promising lead compound for further development of small
β-peptidomimetics against pathogenic Gram-negative bac-
teria. Further studies to address this issue are in progress at
our laboratory.
was evaporated to dryness. The crude β2,2-amino acid methyl
ester (0.35 mmol) was dissolved in a mixture of 1,4-dioxane and
water 5:1 (∼0.35 M) and the pH adjusted to 8 with TEA. Boc2O
(0.42 mmol) dissolved in as little as possible 1,4-dioxane was
added before the solution was stirred at rt for 18 h. The reaction
mixture was acidified to pH 2-3 with 10% citric acid and
extracted three times with ethyl acetate. The organic phase was
dried over MgSO4, filtered, and evaporated to dryness. The
product was used in the following synthesis without any further
purification.
General Procedure for Ester Hydrolysis (GP3) 3a-n. The
synthesis was performed in accordance to Seebach et al.51
In brief, the Boc-protected β2,2-amino acid methyl ester
(0.35 mmol) was dissolved in a mixture of 1,4-dioxane and
water 3:1 (1.17 mM) before lithium hydroxide (2.1 mmol)
dissolved in as little water as possible was added. The reaction
mixture was stirred at reflux under N2 for 18 h before the volume
was reduced to approximately 1/5 under vacuum. Water
(10 mL) was added to the reaction mixture and the pH adjusted
to 1-2 with dropwise addition of 0.1 M HCl. The aqueous
solution was extracted three times with equal volumes of ethyl
acetate. The organic phase was dried over MgSO4, filtered, and
evaporated to dryness. The product was used in the following
synthesis without any further purification.
In conclusion, relatively small structural alterations of the
β-peptidomimetics had a considerable impact on bacterial
strain specificity and an even larger influence on hemolytic
activity. Furthermore, the small size of the β-peptidomimetics
may provide solutions to many common problems associated
with developing AMP’s into novel antimicrobial agents, such
as being cost-efficient to synthesize, more stable against
proteolytic degradation by nature of containing a β2,2-amino
acid derivative, and perhaps more importantly, the small size
of the prepared β-peptidomimetics may involve less risk of
allergic responses. The study has demonstrated that small
β-peptidomimetics can be used to mimic the antimicrobial
potency and selectivity of much larger AMP’s and that such
small amphipathic scaffolds may be promising drug candi-
dates for treatment of serious bacterial infections.
Experimental Section
Chemistry in General. 1H and 13C NMR spectra were re-
corded on 400 or 600 MHz Varian spectrometers. Chemical
General Procedure for Coupling of Boc-Protected β2,2-Amino
Acids and L-Arginine Amide (GP4) 4a-n. The synthesis was
performed in accordance with Chan and White.52 In brief, the
Boc-protected β2,2-amino acid (0.2 mmol) was dissolved in
DMF (0.02 M) and DIPEA (0.6 mmol) was added along with
TFFH (0.2 mmol). The β2,2-amino acid was preactivated for 2 h
shifts are expressed in ppm relative to CHCl3 (1H 7.26 ppm, 13
C
77.0 ppm) or methanol (1H 3.31 ppm, 13C 39.0 ppm). The values
are given in δ 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).
Commercially available compounds and solvents were pur-
chased from Sigma-Aldrich and used without further purifica-
tion. Preparative RP-HPLC was carried out on a Waters system
before H-Arg-NH2 2HCl (0.3 mmol) was added. The reaction
3
mixture was stirred at rt for 7 days before it was diluted with
ethyl acetate and washed with brine. The organic phase was
dried over MgSO4, filtered, and evaporated to dryness. The
crude Boc-protected β-peptidomimetic was deprotected by dis-
solving it in DCM (∼0.4 M) and adding an equivalent volume of
TFA:TIS:water (95:2.5:2.5). The mixture was stirred at rt for 2 h
before it was evaporated to dryness. The crude product was
purified by preparative RP-HPLC. The purity of the pure
β-peptidomimetics was checked by analytical RP-HPLC before
the solution was evaporated to dryness, and the residue was
redissolved in water and lyophilized. All compounds possessed
purity above 95%.
˚
equipped with a RP BondaPak C18, 125 A, 10 μm, 25 mm ꢀ
100 mm column, and eluted with acetonitrile and water, both
containing 0.1% TFA. Analytical HPLC was carried out on a
Waters 2695 HPLC equipped with an RP-HPLC Delta Pak C18,
˚
100 A, 5 μm, 3.9 mm ꢀ 150 mm column and analyzed at
wavelengths 214 and 254 nm with a PDA detector spanning
from wavelengths 210 to 310 nm. All compounds were prepared
by using parallel reaction carousels from Radleys.
Synthesis of Cyano-diallyl Acetic Acid Methyl Ester (1a). The
synthesis was conducted in accordance to GP1. 1H NMR
(CDCl3): δ 2.54-2.58 (2H, m), 2.63-2.68 (2H, m), 3.80 (3H,
s), 5.23 (2H, s), 5.26 (2H, s), 5.78-5.83 (2H, m). 13C NMR
(CDCl3): δ 40.6, 49.3, 53.5, 118.3, 121.0, 130.4, 168.5.
Brown-yellow oil, yield 71%. MS-ESIþ: [M þ H]þ calcd180.1,
found 179.9
General Procedure for Dialkylation of Methyl Cyanoacetate
(GP1) 1a-n. The synthesis was performed in accordance to
Cronin et al.19 In brief, sodium methoxide (20 mmol) was
dissolved in methanol (0.2 M) and methyl cyanoacetate
(20 mmol) was added. The reaction mixture was stirred for
5 min at rt before the desired benzyl bromide (20 mmol) was
added and the solution was heated to reflux. After 15 min, the
solution was cooled to rt. A second portion of sodium meth-
oxide (20 mmol) was added, and after 5 min of stirring at rt, the
second portion of the desired benzyl bromide (20 mmol) was
added followed by 15 min reflux. The volume of the reaction
mixture was reduced to about 1/3 under vacuum and extracted
with water/ethyl acetate. The organic phase was dried over
MgSO4, filtered, and evaporated to dryness. The product was
used in following synthesis without any further purification.
General Procedure for Reduction of Nitriles to Amines with
Ra/Ni and Following Boc-Protection (GP2) 2a-n. The synthesis
was performed in accordance to Cronin et al.19 and Bodanzky
et al.50 In brief, Ra/Ni (approximate 2 mL/g methyl cyano-
acetate derivative was washed 3 times with methanol under argon
before the desired methyl cyanoacetate derivative (3.5 mmol)
dissolved in methanol (0.1 M) was added along with acetic acid
(approximately 1 mL/g methyl cyanoacetate derivative). The
reaction mixture was hydrogenated at 45 °C for 5 days under 1
bar H2 pressure. Afterward, the reaction mixture was filtered
through celite to remove the Ra/Ni before the reaction mixture
Synthesis of Cyano-dicyclohexylmethyl Acetic Acid Methyl
Ester (1b). The synthesis was conducted in accordance to GP1
with the exception of 2 h reflux after each time the bromide was
1
added. H NMR (CDCl3): δ 0.79-1.31 (11H, m), 1.45-1.78
(11H, m), 1.80-1.85 (4H, m), 3.77 (3H, s). 13C NMR (CDCl3): δ
25.9, 31.6, 32.8, 33.7, 40.6, 46.1, 53.0, 119.6, 170.4. Red oil, yield:
45%. MS-ESIþ: [M þ H]þ calcd 292.2, found 292.1
Synthesis of Cyano-di(2-cyclohexyl-eth-1-yl) Acetic Acid
Methyl Ester (1c). The synthesis was conducted in accordance
to GP1 with the exception of 2 h reflux after each time the
1
bromide was added. H NMR (CDCl3): δ 0.83-0.95 (4H, m),
1.08-1.27 (9H, m), 1.32-1.47 (3H, m), 1.59-1.98 (14H, m),
3.69 (3H, s). 13C NMR (CDCl3): δ 26.1, 26.4, 32.7, 32.9, 33.0,
35.1, 37.4, 50.0, 53.1, 119.4, 169.9. Red oil, yield 56%. MS-
ESIþ: [M þ Na]þ calcd 342.2, found 342.1
Synthesis of Cyano-dibenzyl Acetic Acid Methyl Ester (1d).
1
The synthesis was conducted in accordance to GP1. H NMR
(CDCl3): δ 3.16 (2H, d, J=13.6 Hz), 3.38 (2H, d, J=13.2 Hz),
3.59 (s, 3H), 7.36 (m, 10H). 13C NMR (CDCl3): δ 43.3, 53.1,

602 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Hansen et al.
53.4, 118.4, 127.9, 128.6, 129.9, 168.6. Yellow viscous oil, yield
96%. MS-ESIþ: [M þ H]þ calcd 280.1, found 280.0
Synthesis of Cyano-di(3,5-ditert-butyl-benzyl) Acetic Acid
Methyl Ester (1n). The synthesis was conducted in accordance
to GP1. 1H NMR (CDCl3): δ 1.35 (36H, s), 3.15 (2H, d, J=13.3
Hz), 3.38 (2H, d, J=13.3 Hz), 3.51 (3H, s), 7.14-7.40 (6H, m).
13C NMR (CDCl3): δ 31.4, 34.8, 44.0, 52.9, 54.1, 118.6, 122.0,
124.3, 133.2, 150.8, 168.9. White solid product, yield 100%. MS-
ESIþ: [M þ H]þ calcd 504.4, found 504.2.
Synthesis of Cyano-di(2-phenyl-eth-1-yl) Acetic Acid Methyl
Ester (1e). The synthesis was conducted in accordance to GP1
with the exception of 2 h reflux after each time the bromide was
added. 1H NMR (CDCl3): δ 2.12 (2H, dt, J=4.8 Hz, J=13 Hz),
2.28 (2H, m,), 2.45 (2H, m), 2.72 (2H, dt, J=5.6 Hz, J=13.2 Hz),
3.78 (3H, s), 7.24-7.40 (10H, m). 13C NMR (CDCl3): δ 32.5,
36.4, 49.5, 53.3, 118.8, 126.6, 128.5, 128.3, 138.8, 169.1. Red oil,
yield 98%. MS-ESIþ: [M þ H]þ calcd 308.2, found 308.1
Synthesis of Cyano-di(3-phenyl-prop-1-yl) Acetic Acid Methyl
Ester (1f). The synthesis was conducted in accordance to GP1
with the exception of 2 h reflux, after each time the bromide was
added. 1H NMR (CDCl3): δ 1.98 (4H, m), 2.24 (2H, t, J=7.2
Hz), 2.71 (2H, t, J=7.2 Hz), 2.85 (2H, t, J=7.6 Hz), 3.46 (2H, t,
J = 6.8 Hz), 3.83 (3H, s), 7.22-7.36 (10H, m). 13C NMR
(CDCl3): δ 26.9, 35.1, 36.8, 49.5, 53.2, 119.1, 126.1, 128.2,
128.4, 140.7, 169.5. Red oil, yield 100%. MS-ESIþ: [M þ H]þ
calcd 336.2, found 336.1
Synthesis of Boc-2,2-dipropyl β-Amino Acid Methyl Ester (2a).
1
The synthesis was conducted in accordance to GP2. H NMR
(CDCl3): δ 0.84 (6H, t, J = 7.0 Hz), 1.17-1.27 (4H, m),
1.38-1.48 (13H, m), 3.28 (2H, br d, J=6.2 Hz), 3.64 (3H, s).
13C NMR (CDCl3): δ 14.5, 17.2, 28.2, 36.1, 43.5, 50.2, 51.6, 79.0,
155.9, 176.8. Yellow oil, yield 53%. MS-ESIþ: [M þ H]þ calcd
288.2, found 288.1.
Synthesis of Boc-2,2-dicyclohexylmethyl β-Amino Acid Methyl
Ester (2b). The synthesis was conducted in accordance to GP2.
1H NMR (CDCl3): δ 0.82-1.68 (26H, m), 1.48 (9H, s), 3.38 (2H,
d, J=6.3 Hz), 3.63 (3H, s). 13C NMR (CDCl3): δ 26.1, 26.3, 28.3,
33.5, 34.0, 35.2, 43.3, 43.7, 51.4, 79.6, 155.8, 170.0. Yellow oil,
yield 62%. MS-ESIþ: [M þ H]þ calcd 396.3, found 396.2.
Synthesis of Boc-2,2-di(2-cyclohexyl-eth-1-yl) β-Amino Acid
Methyl Ester (2c). The synthesis was conducted in accordance to
GP2. 1H NMR (CDCl3): δ 0.20-0.35 (4H, m), 0.95-1.20 (14H,
m), 1.35-1.40 (9H, m), 1.50-1.68 (12H, m) 3.22 (2H, br d, J=
6.2 Hz), 3.60 (3H, s). 13C NMR (CDCl3): δ 26.1, 26.4, 28.2, 30.9,
31.1, 33.1, 38.0, 43.4, 49.9, 51.5, 78.7, 155.7, 176.9. Yellow oil,
yield 58%. MS-ESIþ: [M þ H]þ calcd 424.3, found 424.5
Synthesis of Boc-2,2-dibenzyl β-Amino Acid Methyl Ester (2d).
Synthesis of Cyano-di(2-methylene-naphthalene) Acetic Acid
Methyl Ester (1g). The synthesis was conducted in accordance to
GP1. 1H NMR (CDCl3): δ 3.33 (2H, d, J=13.5 Hz), 3.51 (3H, s),
3.56 (2H, d, J=13.5 Hz), 7.44 (2H, d, J=8.2 Hz), 7.48-7.49
(4H, m), 7.78 (2H, s), 7.82-7.84 (6H, m). 13C NMR (CDCl3): δ
43.4, 53.2, 53.4, 118.4, 126.1, 126.2, 127.6, 127.7, 127.9, 128.3,
129.1, 131.5, 132.8, 133.3, 168.8. Yellow oil, yield 125%.
MS-ESIþ: [M þ H]þ calcd 380.2, found 379.9.
Synthesis of Cyano-di(4-methyl-benzyl) Acetic Acid Methyl
Ester (1h). The synthesis was conducted in accordance to GP1.
1H NMR (CDCl3): δ 2.38 (6H, s), 3.11 (2H, d, J=13.7 Hz), 3.34
(2H, d, J=13.3 Hz), 3.61 (3H, s), 7.18 (4H, d, J=8.2 Hz), 7.23
(4H, d, J=8.2 Hz). 13C NMR (CDCl3): δ 21.2, 42.9, 53.2, 53.5,
118.6, 129.4, 129.8, 131.1, 137.6, 168.9. Clear oil, yield 103%.
MS-ESIþ: [M þ H]þ calcd 308.2, found 308.1.
1
The synthesis was conducted in accordance to GP2. H NMR
(CDCl3): δ 1.46 (9H, s), 2.81 (2H, d J=13.7 Hz), 3.14 (2H, d, J=
14.0 Hz), 3.29 (2H, br d, J=5.9 Hz), 3.59 (s, 3H), 7.13-7.29
(10H, m). 13C NMR (CDCl3): δ 28.4, 41.3, 43.1, 51.6, 52.2, 79.2,
126.7, 128.2, 130.2, 136.4, 156.0, 175.5. Yellow oil, yield 86%.
MS-ESIþ: [M þ H]þ calcd 384.2, found 384.1.
Synthesis of Boc-2,2-di(2-phenyl-eth-1-yl) β-Amino Acid
Methyl Ester (2e). The synthesis was conducted in accordance
to GP2. 1H NMR (CDCl3): δ 1.55 (9H, s), 1.90-2.00 (4H, m),
2.62-2.71 (4H, m), 3.58 (2H, br d), 3.72 (3H, s), 7.21-7.30 (10H,
m). 13C NMR (CDCl3): δ 28.1, 30.4, 36.0, 43.2, 51.6, 51.8, 79.1,
125.8, 128.2, 128.4, 141.7, 155.9, 176.0. Yellow oil, yield 41%.
MS-ESIþ: [M þ H]þ calcd 412.3, found 412.2.
Synthesis of Cyano-di(4-trifluoromethyl-benzyl) Acetic Acid
Methyl Ester (1i). The synthesis was conducted in accordance to
GP1. 1H NMR (CDCl3): δ 3.17 (2H, d, J=13.5 Hz), 3.40 (2H, d,
J=13.5 Hz), 3.57 (3H, s), 7.43 (4H, d, J=7.6 Hz), 7.61 (4H, d, J=
7.6 Hz). 13C NMR (CDCl3): δ 42.8, 52.8, 53.5, 117.7, 123.9 (q,
J=272.2 Hz), 125.7, 129.3, 130.3, 137.7, 168.0. Yellow oil, yield
133%. MS-ESIþ: [M þ H]þ calcd 416.1, found 416.1
Synthesis of Boc-2,2-di(3-phenyl-prop-1-yl) β-Amino Acid
Methyl Ester (2f). The synthesis was conducted in accordance
to GP2. 1H NMR (CDCl3): δ 1.44 (13H, s), 1.46 (4H, m), 1.54
(4H, m), 2.56 (2H, br t), 3.32 (2H, br d, J=6.2 Hz), 3.65 (3H, s),
7.14-7.29 (10H, m). 13C NMR (CDCl3): δ 25.6, 28.2, 33.2, 36.1,
43.6, 50.0, 51.6, 79.0, 125.7, 128.2, 128.3, 141.8, 155.8, 176.4.
Yellow oil, yield 55%. MS-ESIþ: [M þ H]þ calcd 440.3,
found 440.1.
Synthesis of Cyano-di(4-isopropyl-benzyl) Acetic Acid Methyl
Ester (1j). The synthesis was conducted in accordance to GP1.
1H NMR (CDCl3): δ 1.24 (12H, d, J=6.6 Hz), 2.89 (2H, m), 3.08
(2H, d, J=13.7 Hz), 3.29 (2H, d, J=13.7 Hz), 3.56 (3H, s),
7.17-7.23 (8H, m). 13C NMR (CDCl3): δ 23.9, 33.7, 42.8, 53.1,
53.5, 118.7, 126.6, 129.8, 131.3, 148.4, 168.8. White solid
product, yield 115%. MS-ESIþ: [M þ H]þ calcd 364.2,
found 364.3
Synthesis of Cyano-di(4-tert-butyl-benzyl) Acetic Acid Methyl
Ester (1k). The synthesis was conducted in accordance to GP1.
1H NMR (CDCl3): δ 1.31 (18H, s), 3.28 (2H, d, J=14 Hz), 3.29
(2H, d, J=13.6 Hz), 3.56 (3H, s), 7.22 (4H, d, J=8.4 Hz), 7.34
(4H, d, J=8.4 Hz). 13C NMR (CDCl3): δ 31.3, 34.5, 42.8, 53.1,
53.4, 118.7, 125.5, 129.6, 150.7, 168.8. White viscous, yield 96%.
MS-ESIþ: [M þ H]þ calcd 392.3, found 392.2.
Synthesis of Boc-2,2-di(2-methylene-naphthalene) β-Amino
Acid Methyl Ester (2g). The synthesis was conducted in accor-
dance to GP2. 1H NMR (CDCl3): δ 1.53 (9H, s), 2.98-3.39 (6H,
m), 3.63 (3H, s), 7.26 -7.80 (14H, m). 13C NMR (CDCl3): δ
27.4, 29.3, 51.6, 52.3, 126.0, 127.2, 127.5, 127.7, 128.9, 129.0,
131.0, 133.3. Yellow oil, yield 89%. MS-ESIþ: [M þ H]þ calcd
484.2, found 484.0.
Synthesis of Cyano-di(3,5-dimethyl-benzyl) Acetic Acid Methyl
Ester (1l). The synthesis was conducted in accordance to GP1.
1H NMR (CDCl3): δ 2.39 (12H, s), 3.06 (2H, d, J=13.3 Hz), 3.28
Synthesis of Boc-2,2-di(4-methyl-benzyl) β-Amino Acid Methyl
Ester (2h). The synthesis was conducted in accordance to GP2.
1H NMR (CDCl3): δ 1.44 (9H, s), 2.30 (6H, s), 2.75 (2H, d, J=
14.1 Hz), 3.08 (2H, d, J=14.1 Hz), 3.25 (2H, br d, J=5.9 Hz),
(2H, d, J=13.8 Hz), 3.67 (3H, s), 6.98 (4H, s), 7.02 (2H, s). 13
C
NMR (CDCl3): δ 21.1, 42.9, 52.8, 53.2, 118.3, 127.6, 129.2,
133.8, 137.8, 168.8. Yellow oil, yield 80%. MS-ESIþ: [M þ H]þ
calcd 336.2, found 336.1.
3.68 (3H, s), 7.01 (4H, d, J=8.2 Hz), 7.06 (4H, d, J=7.6 Hz). 13
C
NMR (CDCl3): δ 21.0, 28.4, 40.9, 43.0, 51.6, 52.2, 79.1, 128.9,
130.0, 133.3, 136.2, 156.0, 175.7. Yellow oil, yield 64%. MS-
ESIþ: [M þ H]þ calcd 412.3, found 412.1
Synthesis of Cyano-di(3,5-dimethoxy-benzyl) Acetic Acid
Methyl Ester (1m). The synthesis was conducted in accordance
to GP1. 1H NMR (CDCl3): δ 3.06 (2H, d, J=13.3 Hz), 3.28 (2H,
d, J=13.8 Hz), 3.66 (3H, s), 3.81 (12H, s), 6.44 (2H, s), 6.48 (4H,
s). 13C NMR (CDCl3): δ 43.4, 53.0, 53.3, 55.3, 100.0, 107.9,
118.5, 136.0, 160.7, 168.7. Light-brown viscous oil, yield 100%.
MS-ESIþ: [M þ H]þ calcd 400.2, found 400.1.
Synthesis of Boc-2,2-di(4-trifluoromethyl-benzyl) β-Amino
Acid Methyl Ester (2i). The synthesis was conducted in accor-
dance to GP2 with the exception of only 24 h hydrogenation
with Ra/Ni. 1H NMR (CDCl3): δ 1.35 (9H, s), 3.08 (2H, d, J=
13.3 Hz), 3.20 (2H, br d, J=5.9 Hz), 3.32 (2H, d, J=13.5 Hz),
3.47 (3H, s), 7.17 (2H, d, J=9.7 Hz), 7.33 (2H, d, J=8.1 Hz), 7.44

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 603
Synthesis of Boc-2,2-di(2-phenyl-eth-1-yl) β-Amino Acid (3e).
The synthesis was conducted in accordance to GP3. H NMR
(2H, d, J=7.9 Hz), 7.52 (2H, d, J=8.1 Hz). 13C NMR (CDCl3): δ
28.3, 41.2, 42.8, 51.9, 53.5, 125.4, 125.6, 130.3, 146.7, 168.0.
Yellow oil, yield 98%. MS-ESIþ: [M þ H]þ calcd 520.2, found
520.0.
1
(CDCl3): δ 1.46 (9H, s), 1.90-2.05 (4H, m), 2.60-2.75 (4H, m),
3.54 (2H, d, J = 6.3 Hz), 7.18-7.26 (10H, m). 13C NMR
(CDCl3): δ 28.1, 30.4, 35.9, 43.2, 50.3, 79.6, 125.9, 128.2,
128.4, 141.7, 156.1, 181.7. Yellow solid product, yield 92%.
MS-ESIþ: [M þ H]þ calcd 398.2, found 398.2.
Synthesis of Boc-2,2-di(4-isopropyl-benzyl) β-Amino Acid
Methyl Ester (2j). The synthesis was conducted in accordance
to GP2. 1H NMR (CDCl3): δ 1.20-1.22 (12H, m), 1.45 (9H, s),
2.76 (2H, d, J=13.8 Hz), 2.89 (2H, m), 3.09 (2H, d, J=13.8 Hz),
3.28 (2H, br d, J=6.4 Hz), 3.71 (3H, s), 7.01-7.12 (8H, m).
13C NMR (CDCl3): δ 23.9, 27.4, 33.6, 41.0, 51.6, 51.9, 126.4,
129.9, 132.8, 146.7, 156.3, 172.0. Yellow oil, yield 111%.
MS-ESIþ: [M þ H]þ calcd 468.3, found 468.2.
Synthesis of Boc-2,2-di(3-phenyl-prop-1-yl) β-Amino Acid (3f).
1
The synthesis was conducted in accordance to GP3. H NMR
(CDCl3): δ 1.44 (9H, s), 1.59 (8H, m), 2.57 (4H, br t), 3.33 (2H, br
d, J=6.4 Hz), 7.14-7.29 (10H, m). 13C NMR (CDCl3): δ 25.6,
28.3, 33.1, 36.2, 43.6, 49.8, 79.3, 125.8, 128.2, 128.3, 141.8, 156.0,
182.0. White solid product, yield 58%. MS-ESIþ: [M þ H]þ
calcd 426.3, found 426.5.
Synthesis of Boc-2,2-di(2-methylene-naphthalene) β-Amino
Acid (3g). The synthesis was conducted in accordance to GP3.
1H NMR (CDCl3): δ 1.43 (9H, s), 2.96-3.53 (6H, m), 7.31 -8.07
(14H, m). 13C NMR (CDCl3): δ 28.3, 34.4, 37.9, 49.0, 79.7,
126.0, 127.2, 127.5, 127.5, 127.6, 128.1, 132.2, 133.4, 136.2,
156.0, 180.4. Gray solid product, yield 80%. MS-ESIþ: [M þ
H]þ calcd 470.2, found 470.1.
Synthesis of Boc-2,2-di(4-tert-butyl-benzyl) β-Amino Acid
Methyl Ester (2k). The synthesis was conducted in accordance
1
to GP2. H NMR (CDCl3): δ 1.30 (18H, s), 1.54 (9H, s), 2.77
(2H, d, J=14.0 Hz), 3.11 (2H, d, J=13.3 Hz), 3.30 (2H, br s),
3.69 (3H, s), 7.07 (2H, d, J=7.8 Hz), 7.28 (2H, d, J=7.4 Hz). 13
C
NMR (CDCl3): δ 27.8, 31.3, 34.3, 40.9, 43.2, 51.6, 52.2, 79.0,
125.1, 129.8, 133.3, 149.4, 156.0, 175.7. Yellow oil, yield 75%.
MS-ESIþ: [M þ H]þ calcd 496.3, found 496.2.
Synthesis of Boc-2,2-di(3,5-dimethyl-benzyl) β-Amino Acid
Methyl Ester (2l). The synthesis was conducted in accordance
Synthesis of Boc-2,2-di(4-methyl-benzyl) β-Amino Acid (3h).
The synthesis was conducted in accordance to GP3. H NMR
1
1
to GP2. H NMR (CDCl3): δ 1.47 (9H, s), 2.27 (12H, s), 2.72
(CDCl3): δ 1.47 (9H, s), 2.31 (6H, s), 2.80 (2H, d, J=13.9 Hz),
3.14 (2H, d, J=13.9 Hz), 3.28 (2H, br d, J=6.4 Hz), 7.06-7.12
(8H, m). 13C NMR (CDCl3): δ 21.0, 28.3, 40.8, 42.9, 51.9, 79.3,
128.9, 130.2, 136.2, 156.1, 181.0. Yellow solid product, yield
62%. MS-ESIþ: [M þ H]þ calcd 398.2, found 398.1.
(2H, d, J=13.7 Hz), 3.06 (2H, d, J=16.6 Hz), 3.24 (2H, br s),
3.69 (3H, s), 6.73 (4H, s), 6.85 (2H, s). 13C NMR (CDCl3): δ 21.2,
28.3, 41.0, 42.8, 51.4, 52.0, 79.0, 128.1, 128.3, 136.1, 137.5, 155.9,
175.9. Yellow oil, yield 79%. MS-ESIþ: [M þ H]þ calcd 440.3,
found: 440.1.
Synthesis of Boc-2,2-di(3,5-dimethoxy-benzyl) β-Amino Acid
Methyl Ester (2m). The synthesis was conducted in accordance
to GP2. 1H NMR (CDCl3): δ 1.43 (9H, s), 2.69 (2H, d, J=13.7
Hz), 3.07 (2H, d, J=13.6 Hz), 3.32 (2H, br d, J=5.5 Hz), 3.70
(3H, s), 3.75 (12 H, s), 6.30-6.49 (6H, m). 13C NMR (CDCl3): δ
27.3, 41.3, 42.6, 51.8, 52.1, 55.2, 79.2, 98.9, 108.2, 138.5, 160.5,
175.6. Brown oil, yield 80%. MS-ESIþ: [M þ H]þ calcd 504.2,
found 504.1.
Synthesis of Boc-2,2-di(3,5-di-tert-butyl-benzyl) β-Amino Acid
Methyl Ester (2n). The synthesis was conducted in accordance to
GP2. 1H NMR (CDCl3): δ 1.30 (36H, s), 1.44 (9H, s), 2.80 (2H,
d, J = 13.9 Hz), 3.14 (2H, d, J = 13.8 Hz), 3.25 (2H, br d),
3.42 (3H, s), 7.00-7.36 (6H, m). 13C NMR (CDCl3): δ 27.4,
31.5, 34.7, 44.0, 51.7, 58.2, 122.0, 124.3, 146.7, 150.5, 168.9.
Yellow oil, yield 60%. MS-ESIþ: [M þ H]þ calcd 608.5,
found 608.3
Synthesis of Boc-2,2-di(4-trifluoromethyl-benzyl) β-Amino
Acid (3i). The synthesis was conducted in accordance to GP3.
1H NMR (CDCl3): δ 1.46 (9H, s), 3.17 (2H, d, J=13.5 Hz), 3.28
(2H, br d), 3.42 (2H, d, J=13.5 Hz), 7.39 (2H, d, J=8.1 Hz), 7.52
(2H, d, J=8.1 Hz), 7.57 (2H, d, J=8.1 Hz), 7.63 (2H, d, J=8.1
Hz). 13C NMR (CDCl3): δ 28.2, 37.6, 42.4, 51.7, 80.2, 125.2,
125.8, 129.4, 140.3, 156.4, 179.4. Yellow viscous oil, yield 52%.
MS-ESIþ: [M þ H]þ calcd 506.2, found 506.0.
Synthesis of Boc-2,2-di(4-isopropyl-benzyl) β-Amino Acid (3j).
1
The synthesis was conducted in accordance to GP3. H NMR
(CDCl3): δ 1.25 (12H, t, J=5.7 Hz), 1.47 (9H, s), 2.81 (2H, d, J=
13.0 Hz), 2.88 (2H, m), 3.17 (2H, d, J=13.9 Hz), 3.31 (2H, br d,
J=5.4 Hz), 7.14-7.16 (8H, m). 13C NMR (CDCl3): δ 23.9, 28.4,
33.6, 40.8, 49.0, 51.9, 79.8, 126.4, 129.9, 132.8, 146.7, 156.3,
172.0. Yellow solid product, yield 62%. MS-ESIþ: [M þ H]þ
calcd 454.3, found 454.1.
Synthesis of Boc-2,2-di(4-tert-butyl-benzyl) β-Amino Acid
(3k). The synthesis was conducted in accordance to GP3. H
Synthesis of Boc-2,2-dipropyl β-Amino Acid (3a). The synth-
esis was conducted in accordance to GP3. 1H NMR (CDCl3): δ
0.90 (6H, m), 1.17-1.31 (4H, m), 1.42 (9H, s), 1.51-1.53 (4H,
m), 3.32 (2H, br d, J=6.3 Hz). 13C NMR (CDCl3): δ 14.5, 17.2,
28.3, 36.0, 43.5, 50.1, 79.2, 156.0, 182.6. Yellow solid product,
yield 75%. MS-ESIþ: [M þ H]þ calcd 274.2, found 274.1.
Synthesis of Boc-2,2-dicyclohexylmethyl β-Amino Acid (3b).
1
NMR (CDCl3): δ 1.25 (27H, s), 2.78 (2H, br s), 3.13 (2H, br s),
7.13 (4H, br s), 7.23 (4H, br s). 13C NMR (CDCl3): δ 28.3, 31.3,
34.3, 40.9, 51.5, 79.3, 125.0, 130.1, 133.3, 149.2, 156.3, 181.7.
White solid product, yield 56%. MS-ESIþ: [M þ H]þ calcd
482.3, found 482.1.
1
Synthesis of Boc-2,2-di(3,5-dimethyl-benzyl) β-Amino Acid
(3l). The synthesis was conducted in accordance to GP3. H
The synthesis was conducted in accordance to GP3. H NMR
1
(CDCl3): δ 0.84-1.63 (26H, m), 1.42 (9H, s), 3.38 (2H, d, J=6.2
Hz). 13C NMR (CDCl3): δ 26.1, 26.4, 28.3, 33.6, 35.3, 43.4, 48.4,
51.4, 79.2, 156.0, 182.5. Yellow solid product, yield 17%. MS-
ESIþ: [M þ H]þ calcd 382.3, found 382.2.
NMR (CDCl3): δ 1.46 (9H, s), 2.25 (12 H, s), 2.74 (2H, d, J=12.9
Hz), 3.09 (2H, d, J=12.3 Hz), 3.24 (2H, br s), 6.80 (2H, s), 6.85
(1H, s). 13C NMR (CDCl3): δ 21.2, 28.4, 40.9, 42.8, 51.9, 79.3,
126.7, 128.3, 135.9, 137.5, 156.1, 181.6. White solid product,
yield 70%. MS-ESIþ: [M þ H]þ calcd 426.3, found 426.2.
Synthesis of Boc-2,2-di(3,5-dimethoxy-benzyl) β-Amino Acid
Synthesis of Boc-2,2-di(2-cyclohexyl-eth-1-yl) β-Amino Acid
1
(3c). The synthesis was conducted in accordance to GP3. H
NMR (CDCl3): δ 0.80-0.90 (4H, m), 1.08-1.25 (14H, m), 1.42
(9H, s), 1.50-1.72 (12H, m), 3.32 (2H, br d). 13C NMR (CDCl3):
δ 26.2, 26.3, 28.3, 31.1, 33.1, 33.2, 38.2, 43.5, 49.8, 79.2, 155.9,
182.4. Yellow solid product, yield 100%. MS-ESIþ: [M þ H]þ
calcd 410.3, found 410.3.
Synthesis of Boc-2,2-dibenzyl β-Amino Acid (3d). The synth-
esis was conducted in accordance to GP3. 1H NMR (CDCl3): δ
1.46 (9H, s), 2.83 (2H, d J=14.1 Hz), 3.17 (2H, d, J=13.5 Hz),
3.29 (2H, br s), 7.20-7.28 (10H, m). 13C NMR (CDCl3): δ 28.4,
37.7, 41.3, 51.9, 79.4, 126.8, 128.2, 128.5, 136.2, 156.2, 180.9.
Off-white solid product, yield 76%. MS-ESIþ: [M þ H]þ calcd
370.2, found 370.2.
1
(3m). The synthesis was conducted in accordance to GP3. H
NMR (CDCl3): δ 1.44 (9H, s), 2.71 (2H, d, J=13.7 Hz), 3.11
(2H, d, J=13.6 Hz), 3.32 (2H, br d, J=5.5 Hz), 3.73 (12 H, s),
6.31-6.49 (6H, m). 13C NMR (CDCl3): δ 28.3, 41.2, 42.6, 55.2,
55.3, 58.0, 79.4, 99.7, 105.2, 108.3, 140.9, 160.4, 180.7. Brown
solid product, yield 55%. MS-ESIþ: [M þ H]þ calcd 490.2,
found 490.1.
Synthesis of Boc-2,2-di(3,5-di-tert-butyl-benzyl) β-Amino Acid
(3n). The synthesis was conducted in accordance to GP3. H
1
NMR (CDCl3): δ 1.30-1.40 (45 H, m), 3.12 (2H, d, J=13.5 Hz),
3.38 (2H, d, J=13.5 Hz), 3.31 (2H, br d), 7.03-7.41 (6H, m).

604 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Hansen et al.
13C NMR (CDCl3): δ 28.4, 31.5, 34.7, 53.8, 58.1, 79.0, 120.5,
122.0, 124.6, 136.9, 150.5, 156.0, 182.6. Yellow solid product,
yield 23%. MS-ESIþ: [M þ H]þ calcd 594.5, found 594.3.
Synthesis of 2,2-Dipropyl β-Ala-Arg-NH2 (4a). The synthesis
42.0, 43.8, 50.4, 54.4, 54.5, 130.3, 131.3, 133.4, 138.3, 158.7,
175.9, 176.2. HRMS-ESIþ: [M þ H]þ calcd 453.2978, found
453.2984.
Synthesis of 2,2-Di(4-trifluoromethyl-benzyl) β-Ala-Arg-NH2
(4i). The synthesis was conducted in accordance to GP4. H
1
1
was conducted in accordance to GP4. H NMR (CD3OD): δ
NMR (CD3OD): δ 1.65-1.76 (2H, m), 1.80-1.84 (1H, m),
1.91-1.98 (1H, m), 2.96-3.03 (2H, m), 3.11-3.27 (2H, m),
3.26 (2H, t, J=7.2 Hz), 4.43 (1H, t, J=7.3 Hz), 7.46-7.51 (4H,
m), 7.66-7.70 (4H, m). 13C NMR (CD3OD): δ 26.7, 30.0, 40.4,
42.0, 43.6, 50.3, 54.7, 108.8, 126.6, 132.2, 158.7, 174.9, 176.2.
HRMS-ESIþ: [M þ H]þ calcd 561.2413, found 561.2420.
Synthesis of 2,2-Di(4-isopropyl-benzyl) β-Ala-Arg-NH2 (4j).
0.93 (6H, dt, J = 2.83 Hz, J = 7.21), 1.22-1.33 (4H, m),
1.60-1.71 (6H, m), 1.77-1.81 (1H, m), 1.87-1.90 (1H, m),
3.10 (2H, s), 3.22 (2H, dt, J=2.3 Hz, J=7.1 Hz), 4.40 (1H, dd, J=
5.4 Hz, J=9.2 Hz). 13C NMR (CD3OD): δ 14.7, 18.0, 18.2, 26.7,
29.9, 36.8, 37.0, 41.9, 43.8, 54.2, 158.7, 176.6, 177.3. HRMS-
ESIþ: [M þ H]þ calcd 329.2665, found 329.2658.
Synthesis of 2,2-Di(2-cyclohexyl-eth-1-yl) β-Ala-Arg-NH2
1
1
(4b). The synthesis was conducted in accordance to GP4. H
The synthesis was conducted in accordance to GP4. H NMR
(CD3OD): δ 1.23 (12H, dd, J=1.5 Hz, J=6.9 Hz), 1.63-1.74
(2H, m), 1.79-1.84 (1H, m), 1.89-1.94 (1H, m), 2.87-3.00 (6H,
m), 3.18-3.25 (4H, m), 4.41 (1H, dd, J=6.4 Hz, J=8.1 Hz),
7.13-7.21 (8H, m). 13C NMR (CD3OD): δ 24.4, 26.6, 30.2, 35.0,
40.6, 42.0, 43.8, 50.5, 54.5, 127.7, 131.5, 133.7, 133.9, 149.3,
158.7, 176.0, 176.2. HRMS-ESIþ: [M þ H]þ calcd 509.3604,
found 509.3610.
NMR (CD3OD): δ 0.98-1.14 (4H, m), 1.16-1.20 (2H, m),
1.23-1.35 (6H, m), 1.57-1.78 (18H, m), 1.85-1.89 (1H, m),
3.17 (2H, s), 3.21 (2H, dt, J=2.3 Hz, J=7.1 Hz), 4.39 (1H, dd, J=
5.9 Hz, J=8.3 Hz). 13C NMR (CD3OD): δ 26.7, 27.1, 27.4, 30.1,
34.9, 35.7, 36.0, 36.3, 41.9, 42.2, 42.8, 44.7, 48.4, 54.2, 158.6,
176.3, 177.7. HRMS-ESIþ: [M þ H]þ calcd 437.3604, found
437.3608.
Synthesis of 2,2-Di(4-tert-butyl-benzyl) β-Ala-Arg-NH2 (4k).
The synthesis was conducted in accordance to GP4. H NMR
Synthesis of 2,2-Di(2-cyclohexyl-eth-1-yl) β-Ala-Arg-NH2
(4c). The synthesis was conducted in accordance to GP4. H
1
1
(CD3OD): δ 1.31 (18H, s), 1.62-1.77 (2H, m), 1.77-1.86 (1H,
m), 1.88-1.95 (1H, m), 2.94-3.00 (4H, m), 3.18-3.32 (4H, m),
4.42 (1H, t, J=7.2 Hz), 7.16 (4H, dd, J=8.2 Hz, J=17.3 Hz) 7.37
(4H, dd J=3.4 Hz, J=8.2 Hz). 13C NMR (CD3OD): δ 26.7, 30.2,
31.7, 35.3, 42.1, 43.8, 50.5, 54.5, 54.6, 126.6, 131.2, 133.5, 151.5,
158.7, 176.0, 176.3. HRMS-ESIþ: [M þ H]þ calcd 537.3917,
found 539.3907.
NMR (CD3OD): δ 0.92-1.00 (4H, m), 1.12-1.32 (12H, m),
1.64-1.84 (17H, m), 1.89-1.94 (1H, m), 3.12 (2H, s), 3.25 (2H,
dt, J=3.1 Hz, J=7.1 Hz), 4.44 (1H, dd, J=5.3 Hz, J=9.2 Hz).
13C NMR (CD3OD): δ 26.8, 27.4, 27.7, 29.9, 31.8, 31.8, 32.1,
32.3, 34.3, 34.4, 34.6, 39.4, 39.5, 41.9, 44.0, 54.2. 158.6,
176.6, 177.3. HRMS-ESIþ: [M þ H]þ calcd 465.3604, found
465.3608.
Synthesis of 2,2-Di(3,5-dimethyl-benzyl) β-Ala-Arg-NH2 (4l).
The synthesis was conducted in accordance to GP4. H NMR
Synthesis of 2,2-Dibenzyl β-Ala-Arg-NH2 (4d). The synthesis
was conducted in accordance to GP4. H NMR (CD3OD): δ
1
1
(CD3OD): δ 1.58-1.73 (2H, m), 1.75-1.82 (1H, m), 1.86-1.95
(1H, m), 2.28 (12H, d, J=6.6 Hz), 2.9-3.00 (4H, m), 3.13 (2H,
dd J=3.6 Hz, J=14.3 Hz), 3.22 (2H, dt, J=2.7 Hz, J=7.0 Hz),
1.56-1.72 (2H, m), 1.76-1.84 (1H, m), 1.86-1.94 (1H, m),
2.94-3.03 (4H, m), 3.20-3.25 (4H, m), 4.41 (1H, t, J=7.0 Hz),
7.22-7.35 (10H, m). 13C NMR (CD3OD): δ 26.7, 30.1, 41.0,
41.9, 43.8, 50.4, 54.4, 54.6, 128.4, 129.6, 131.5, 136.5, 158.7,
175.7, 176.3. HRMS-ESIþ: [M þ H]þ calcd 425.2665, found
425.2652.
4.42 (1H, t J=7.2 Hz), 6.94 (4H, s), 6.92 (1H, s), 6.92 (1H, s). 13
C
NMR (CD3OD): δ 21.3, 26.7, 30.4, 40.1, 42.1, 44.1, 50.2, 54.4,
129.1, 129.9, 136.4, 139.4, 158.7, 175.9, 176.1. HRMS-ESIþ: [M
þ H]þ calcd 481.3291, found 481.3302.
Synthesis of 2,2-Di(2-phenyl-eth-1-yl) β-Ala-Arg-NH2 (4e).
The synthesis was conducted in accordance to GP4. H NMR
1
Synthesis of 2,2-Di(3,5-dimethoxy-benzyl) β-Ala-Arg-NH2
1
(4m). The synthesis was conducted in accordance to GP4. H
(CD3OD): δ 1.68-1.86 (3H, m), 1.91-1.97 (1H, m), 2.07-2.16
(4H, m), 2.60-2.76 (4H, m), 3.25 (2H, t, J=7.1 Hz), 4.50 (1H, q,
J = 5.6 Hz, J = 9.0 Hz), 7.21-7.33 (10H, m). 13C NMR
(CD3OD): δ 26.9, 29.8, 36.5, 36.9, 41.9, 43.8, 54.4, 127.3,
129.5, 129.6, 142.5, 176.6. HRMS-ESIþ: [M þ H]þ calcd
453.2978, found 453.2986.
NMR (CD3OD): δ 1.60-1.72 (2H, m), 1.77-1.83 (1H, m),
1.87-1.94 (1H, m), 2.94-3.07 (4H, m), 3.14-3.23 (4H, m),
3.75, 3.76 (12H, 2s), 4.44 (1H, dd, J = 5.7 Hz, J = 8.5 Hz),
6.37-6.43 (6H, m). 13C NMR (CD3OD): δ 26.6, 30.4, 41.1, 41.3,
42.0, 44.3, 50.1, 54.5, 55.8, 99.8, 100.2, 109.4, 109.7, 138.7, 138.8,
158.7, 162.5, 175.9, 176.4. HRMS-ESIþ: [M þ H]þ calcd
545.3088, found 545.3101.
Synthesis of 2,2-Di(3-phenyl-prop-1-yl) β-Ala-Arg-NH2 (4f).
The synthesis was conducted in accordance to GP4. H NMR
1
Synthesis of 2,2-Di(3,5-di-tert-butyl-benzyl) β-Ala-Arg-NH2
(4n). The synthesis was conducted in accordance to GP4. H
(CD3OD): δ 1.46-1.78 (11H, m), 1.84-1.89 (1H, m), 2.57-2.65
(4H, m), 3.09 (2H, s), 3.20 (2H, t, J=7.0 Hz), 4.38 (1H, m),
7.14-7.18(6H, m), 7.24-7.28 (4H, m). 13C NMR (CD3OD): δ
26.8, 29.9, 33.8, 36.9, 42.0, 44.1, 46.3, 54.3, 127.1, 129.3, 129.5,
143.0, 158.7, 176.6, 177.0. HRMS-ESIþ: [M þ H]þ calcd
481.3291, found 481.3297.
1
NMR (CD3OD): δ 1.31 (36H, m), 1.64-1.72 (2H, m), 1.79-1.86
(1H, m), 1.92-1.96 (1H, m), 2.88-3.04 (3H, m), 3.15-3.24 (4H,
m), 4.44 (1H, dd, J=5.8 Hz, J=8.4 Hz), 7.05 (2H, br d, J=1.6
Hz), 7.09 (2H, br d, J=1.6 Hz), 7.37 (2H, br d, J=1.9 Hz). 13C
NMR (CD3OD): δ 26.8, 30.4, 31.9, 35.7, 40.4, 40.6, 42.0, 44.4,
49.7, 54.8, 122.2, 122.3, 125.5, 125.7, 135.8, 136.0, 152.3, 153.4,
158.7, 176.5, 176.6. HRMS-ESIþ: [M þ H]þ calcd 649.5169,
found 649.5161.
Antimicrobial Activity. The antimicrobial testing was con-
ducted by TosLab A/S (Tromsø, Norway). Each compound was
diluted to 1 mg/mL in water and tested in duplicates at 200, 100,
50, 35, 15, 10, 5, 2.5, 1, and 0.5 μg/mL, except for compound 4n,
which was tested at 50, 35, 15,10, 5, 2.5, 1, and 0.5 μg/mL due to
limited water solubility. All tested β-peptidomimetics were di-
TFA salts. All compounds displaying MIC values below 50 μg/
mL were retested using the earlier stated procedure.
Synthesis of 2,2-Di(2-methylene-naphthalene) β-Ala-Arg-NH2
(4g). The synthesis was conducted in accordance to GP4. H
1
NMR (CD3OD): δ 1.53-1.64 (2H, m), 1.75-1.82 (1H, m),
1.86-1.94 (1H, m), 3.06-3.15 (4H, m), 3.22-3.29 (2H, m),
3.45-3.51 (2H, m), 4.45 (1H, dd, J = 6.3 Hz, J = 8.3 Hz),
7.36-7.40 (2H, m), 7.45-7.51 (4H, m), 7.75 (1H, s), 7.78 (1H, s),
7.82-7.87 (6H, m). 13C NMR (CD3OD): δ 26.6, 30.2, 41.1, 41.2,
42.0, 44.1, 50.9, 54.6, 127.1, 127.2, 127.4, 127.5, 128.6, 128.7,
128.9, 129.3, 129.4, 130.3, 130.4, 134.0, 134.1, 134.8, 134.9,
158.6, 175.8, 176.3. HRMS-ESIþ: [M þ H]þ calcd 525.2978,
found 525.3002.
Synthesis of 2,2-Di(4-methyl-benzyl) β-Ala-Arg-NH2 (4h).
The synthesis was conducted in accordance to GP4. H NMR
1
Hemolytic Activity. For the RBC toxicity tests, 8 mL of blood
was collected from healthy adult male donors. The blood was
divided equally and distributed into a commercial available
EDTA containing test tube (BD vacutainer, 7.2 mg K2 EDTA)
and into a 10 mL reaction vial containing 40 μL of a heparin
(CD3OD): δ 1.60-1.74 (2H, m), 1.78-1.86 (1H, m), 1.86-1.94
(1H, m), 2.35 (6H, d, J=0.3 Hz), 2.94-3.01 (4H, m), 3.20 (2H, d,
J=14.4 Hz), 3.25 (2H, t, J=7.1 Hz), 4.43 (1H, t, J=7.2 Hz),
7.12-7.18 (8H, m). 13C NMR (CD3OD): δ 21.1, 26.6, 30.1, 40.5,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 605
solution (1000 U/mL in 0.9% sodium chloride). After 30 min,
the hematocrit of the EDTA treated blood was determined. The
heparinized blood was centrifuged for 10 min at 1500 rpm and
the supernatant removed. Subsequently, the RBCs were washed
with prewarmed PBS three times and diluted to 10% hemato-
crit. The β-peptidomimetics were dissolved in PBS (concen-
tration 1 μg/mL to 1000 μg/mL) and incubated together with
the RBCs under agitation at 37 °C for 1 hour. A positive control
with an end concentration of 0.1% Triton X-100 and a negative
control containing pure PBS buffer were included. The samples
were centrifuged (4000 rpm) for 5 min, and the absorption of the
supernatant was measured at 405 nm. The percentage of hemo-
lysis was calculated as the ratio of the peptide treated sample and
the Triton X-100 treated sample. Each compound was tested
from a concentration of 1000 μg/mL and lower, except for
compound 4k, which was only tested up to 500 μg/mL, and
compound 4n, which was only tested up to 71.5 μg/mL due to
limited solubility in the PBS buffer. The values given in Table 1
correspond to 20% hemolysis (EC20).
Table 2. Optimized MS Conditions for External Standard and the
Different β-Peptidomimetics Undergoing Enzymatic Degradation
collision
compd ion transition energy (eV) voltage (V) window (min)
cone
retention
4c
4d
4g
4i
233.2 f 174.1
213.1 f 174.1
263.2 f 141.3
281.1 f 174.3
269.2 f 174.2
327.1 f 174.1
13.00
18.00
23.00
14.00
15.00
18.00
26.00
24.00
18.00
26.00
22.00
29.00
21.00
36.00
4.0-10.0
5.0-10.0
5.0-10.0
5.0-10.0
5.0-10.0
5.0-10.0
2.0-5.0
4k
4m
atenolol (ES)267.1 f 145.0
purified water with 5% acetonitrile and 0.1% trifluoro acetic
acid, and solvent B, 95% acetonitrile, 5% purified water, and
0.1% trifluoro acetic acid. The gradient used on this system
started with an isocratic elution of 100% A for 3 min and was
then increased linearly to 95% B after 33 min. Detection was
performed with a Waters 996 PDA detector at 350 nm, which
was appropriate for detection of the para-nitroaniline moiety.
No internal standard was used; the degree of hydrolysation was
determined based on relative ratios of the two peaks corre-
sponding to peptide and the para-nitroaniline group. The flow
rate was 1 mL/min at all times.
Stability. The stability testing against R-chymotrypsin was
conducted by dissolving the β-peptidomimetics (1 mg/mL) in
water. R-Chymotrypsin was dissolved (0.1 mg/mL) in 1 mM
HCl containing 2 mM CaCl2. The enzymatic digestion was
performed in 100 mM TRIS HCl buffer containing 10 mM
CaCl2. Final enzyme concentration was 2 μg/mL, and final
β-peptidomimetic concentration was 100 μg/mL in a total
volume of 0.5 mL. Then 15 μL samples were collected at 0, 15,
30, 60, 120, and 240 min, in addition to samples collected at 24
and 48 h. An external standard (atenolol hydrochloride) was
applied to the sample together with 100 μL 10% acetic acid to
terminate enzymatic digestion before the sample was diluted to 1
mL with water. For every test, a negative control without
enzyme was incubated to ensure that degradation was due to
enzyme activity and not to other factors. Succinyl-Ala-Ala-Pro-
Phe-para-nitroanhiline was used as positive control. All tests
were run in triplicates. Quantitative analyses of remaining
β-peptidomimetics were done by HPLC-MS. HPLC separation
was done with a Waters 2695 separation module with a Sunfire
Acknowledgment. We thank Espen Hansen (MarBio,
Tromsø) for invaluable help with HRMS and Lytix
Biopharma AS for financial support.
Supporting Information Available: MBC values and purity
data for compounds undergoing biological evaluation. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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