Synthesis and lipophilicity evaluation of some novel indole-containing pseudopeptides

Article abstract of DOI:10.1007/s00706-013-1098-0

An efficient synthesis of indole-containing pseudopeptides via an Ugi four-component reaction among indolecarboxylic acids, aromatic aldehydes, primary amines, and isocyanides in methanol is described. The lipophilicity of the synthesized compounds was determined using the n-octanol/PBS shake-flask procedure. The lipophilicity studies showed that most of the compounds possess the optimal range of lipophilicity for oral absorption, cell membrane penetration, and blood-brain barrier permeation. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-013-1098-0

Monatsh Chem (2014) 145:349–356
DOI 10.1007/s00706-013-1098-0
ORIGINAL PAPER
Synthesis and lipophilicity evaluation of some novel
indole-containing pseudopeptides
•
Nahid S. Alavijeh Sorour Ramezanpour Mohammad S. Alavijeh
•
•
•
Saeed Balalaie Frank Rominger Anil Misra Hamid Reza Bijanzadeh
•
•
Received: 24 July 2013 / Accepted: 3 October 2013 / Published online: 5 November 2013
Ó Springer-Verlag Wien 2013
Abstract An efficient synthesis of indole-containing
pseudopeptides via an Ugi four-component reaction among
indolecarboxylic acids, aromatic aldehydes, primary
amines, and isocyanides in methanol is described. The
lipophilicity of the synthesized compounds was determined
using the n-octanol/PBS shake-flask procedure. The lipo-
philicity studies showed that most of the compounds
possess the optimal range of lipophilicity for oral absorp-
tion, cell membrane penetration, and blood-brain barrier
permeation.
used as drugs to assist in treatment of human immune and
neurological disorders [1–3]. Among the large family of
indoles, indolecarboxamides have recently attracted a great
deal of attention from chemists and biologists largely
because of their various pharmacological activities such as
antioxidant [4], hypocholestrolemic [5], antihistaminic [6],
and so on [7–9].
In recent years, peptides, especially those containing
tryptophan, have gained popularity as promising building
blocks for the design and development of novel materials
with potential application in diverse areas ranging from
drug design to biotechnology [10–12]. Despite their wide
range of bioactivities, polypeptides are generally consid-
ered as poor drugs because of their short half-life, rapid
metabolism, and poor oral bioavailability. Furthermore, the
larger and more flexible the molecule, the more likely it is to
interact with a wide range of receptors with corresponding
loss of specificity. Nevertheless, to overcome these prob-
lems, pharmacokinetic properties of peptides can be
improved by different types of modification such as pepti-
domimetic modification or cyclization of linear peptides
[13]. Among peptidomimetic approaches, pseudopeptides
(compounds containing two or more amide bonds) are
particularly attractive [14].
Keywords Indole-containing pseudopeptides Á
Ugi four-component reaction Á Lipophilicity
Introduction
The indole nucleus is a substructure found in numerous
natural products and pharmaceuticals possessing anti-
inflammatory, antimalarial, antidepressant, anticancer, and
various other activities. It was found that indole com-
pounds, derivatives, and complexes have the potential to be
N. S. Alavijeh Á S. Ramezanpour Á S. Balalaie (&) Á
H. R. Bijanzadeh
Peptide Chemistry Research Center,
K. N. Toosi University of Technology,
P.O. Box 15875-4416, Tehran, Iran
e-mail: balalaie@kntu.ac.ir
Although functionalized indole ring systems have been
found frequently in biologically active compounds, indole
derivatives as multi-component reaction (MCR) partners,
flexible reactions for the rapid generation of complex mol-
ecules with often biologically relevant scaffold structures
[15–17], are rather under-represented [18]. Subsequently,
in continuation of our studies on the construction of active
molecules [19–24], we have been greatly encouraged to
synthesize some novel indoles containing pseudopeptides
through MCRs. Among the protocols for the preparation
of pseudopeptide derivatives, the Ugi four-component
M. S. Alavijeh Á A. Misra
Pharmidex Pharmaceutical Services Ltd., 3rd Floor,
14 Hanover Street, Mayfair, London W1S 1YH, UK
F. Rominger
Organisch-Chemisches Institut der Universitat Heidelberg,
¨
Im Neuenheimer Feld 270, 69120 Heidelberg, Germany
123

350
N. S. Alavijeh et al.
activities and drug design, the lipophilicity of the synthe-
sized compounds was measured using the n-octanol/
phosphate-buffered saline (PBS) shake-flask procedure,
and we proposed that it may represent an important factor
governing their blood-brain barrier as well as cell mem-
brane penetration.
Scheme1
Results and discussion
In the present study, we synthesized a series of novel
indole-containing pseudopeptides by an Ugi-4CR strategy
via the reaction of benzaldehyde derivatives, primary
amines, indolecarboxylic acids, and isocyanides, which
selectively precipitated the desired products out of the
reaction mixtures (Table 1).
reaction offers significant advantages, especially with
respect to diversity, complexity, green chemistry, and atom
economy [25, 26]. In this approach to insert indole units in
the pseudopeptide backbones, indolecarboxylic acids were
used as our Ugi acidic partners because of their known
bioactivities [2] (Scheme 1).
The structures of the synthesized compounds were ver-
ified from their IR, 1H and 13C NMR, and HRMS data. For
instance, the 1H NMR spectra of compound 5a displayed a
singlet for HC (sp3) at 6.17 ppm and also a broad singlet at
5.90 for the amide NH. The 13C NMR spectrum revealed
two distinct peaks at 162.4 and 168.9 ppm for the amide
C=O groups. The structure of the product was confirmed by
using X-ray crystallographic data. Figure 1 shows the
crystal structure of compound 5d.
The relative hydrophilic and hydrophobic properties of
the drug-like compounds are critically important, affecting
their absorption, distribution, metabolism, and excretion
(ADME properties), as well as their solubility, pharmaco-
dynamic and toxicological profile [27]. Usually, log D at
pH 7.4 is a better descriptor for lipophilicity and gives
more physiologically relevant results, instead of the parti-
tion coefficient [28]. The property log D7.4 takes into
account the ionization state of the molecule, important
because many drug-like compounds will be ionized at
physiological pH [29]. Since lipophilicity as a physico-
chemical parameter plays an important role in biological
This synthetic approach permitted the introduction of
four points of diversity within the resulting scaffolds, thus
generating a small library of indole-containing pseudo-
peptides. In most cases, the Ugi reaction occurred
smoothly, affording the desired purified products in satis-
factory yields. It is evident from Table 1 that the yields
decreased by using 2-bromo- and 2-iodoanilines because of
increasing steric hindrance at the functional groups.
Table 1 Synthesized indole-containing pseudopeptides via an Ugi four-component reaction
Entry
Aldehyde
Amine
Acid
Isocyanide
Product
Yield/%
1
Ph
2-Br-C6H4
2-I-C6H4
PhCH2
Indole-2
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
c-Hex
t-Bu
c-Hex
t-Bu
t-Bu
t-Bu
c-Hex
t-Bu
t-Bu
5a
5b
5c
5d
5e
5f
60
40
75
54
70
76
85
65
82
60
50
70
72
65
45
2
Ph
Indole-2
3
Ph
Indole-2
4
Indole-3
4-Cl-C6H4
4-Cl-C6H4
4-Cl-C6H4
4-Br-C6H4
4-Br-C6H4
Ph
2-Br-C6H4
PhCH2
Indole-2
5
Indole-2
6
PhCH2
Indole-3
7
PhCH2
Indole-3
5g
5h
5i
8
PhCH2
Indole-3
9
PhCH2
Indole-3
10
11
12
13
14
15
2-Br-C6H4
2-I-C6H4
PhCH2
Indole-3-CH2
Indole-3-CH2
Indole-3-CH2
Indole-3-CH2
Indole-3-C2H4
Indole-3-C2H4
5j
Ph
5k
5l
4-Cl-C6H4
4-Cl-C6H4
Ph
PhCH2
5m
5n
5o
2-Br-C6H4
2-I-C6H4
Ph
123

Indole-containing pseudopeptides
351
Fig. 1 ORTEP drawing of compound 5d
Lipophilicity is the tendency of a compound to parti-
tion between lipophilic organic phase (immiscible with
water—hydrophobic) and the polar aqueous phase
(hydrophilic). A drug’s distribution (D) coefficient is one
of the most significant physical–chemical properties. It has
an important bearing not only on absorption, distribution,
metabolism, and excretion (ADME) properties, but also
the potency, duration, and mechanism of action of drugs.
Log D coefficients are the ratio of concentrations of a
compound in the two phases of a mixture of two immis-
cible solvents at equilibrium. Ideally, a drug candidate
should have a degree of lipophilicity (log D value from 0
to 3) for improved solubility, permeability, and
absorption.
Fig. 2 Lipophilicity of the synthesized indole-containing pseudo-
peptides 5a–5o
The lipophilicity of the 15 new indole-containing
pseudopeptides was determined using the n-octanol/PBS
shake-flask procedure. The shake-flask method involves
dissolving the compound in a known volume of octanol
(hydrophobic) and PBS (hydrophilic), then measuring the
concentration of the compound in each phase. The prop-
erties of octanol (i.e. hydrophobic) resemble those of lipid
bilayer membranes on cells. Hydrophobic drugs (high
partition coefficients) are preferentially distributed to
hydrophobic compartments of cells such as the lipid
bilayers. Compounds with higher lipophilicity have higher
permeation across biological membranes. Hydrophilic
drugs (low partition coefficients) are found in hydrophilic
compartments such as blood serum.
Known lipophilic compounds were ketoconazole (anti-
fungal agent), propranolol (beta blocker), and verapamil
(calcium channel antagonist). Known hydrophilic com-
pounds were tetracycline (antibiotic) and atenolol (beta
blocker).
The relationship between log D and permeation is non-
linear, with permeation decreasing in both the low and high
ends of log D values, and an optimal range for lipophilicity
tends to be a log D value between 0 and 3 [30]. Typically,
these compounds have a good balance between solubility
and permeability, and this range tends to be optimal
for oral absorption and cell membrane penetration. Com-
pounds with a higher lipophilicity have an increased
metabolism and poor absorption, and a high probability of
binding to unwanted hydrophobic macromolecules, hence
having an increased potential for toxicity [31]. It is evident
from Fig. 2 that all the synthesized indole-containing
pseudopeptides have the optimal range of lipophilicity for
oral absorption, cell membrane penetration, and blood-
brain barrier permeation.
Compound levels in both layers are quantified using an
LC–MS/MS. Results are expressed as log D (pH 7.4) from
the equation log D (pH 7.4) = log10 (peak area for n-oct-
anol sample/peak area for buffer sample). The results are
shown in Fig. 2.
Five compounds were used as internal controls for
lipophilicity. These compounds were selected on the basis
of molecular diversity and with a range of log D values.
123

352
N. S. Alavijeh et al.
168.9 ppm; HR-ESI–MS: calc. for C27H27BrN3O2
([M ? 1]?) 504.12839, found 504.12846.
Conclusions
In conclusion, herein we have reported the synthesis of
some new lipophilic indole-containing pseudopeptides via
Ugi-4CR. All the synthesized indole-containing pseudo-
peptides possess the optimal range of lipophilicity for oral
absorption, cell membrane penetration, and blood-brain
barrier permeation. These compounds have a huge potential
for further reactions and also complexations. Furthermore,
due to the biological importance of the indole nucleus and
presence in central nervous system (CNS) drugs, numerous
studies are underway at this time to explore their anticon-
vulsant, antidepressant, and anti-inflammatory activities.
N-[(tert-Butylcarbamoyl)(phenyl)methyl]-N-
(2-iodophenyl)-1H-indole-2-carboxamide
(5b, C27H26IN3O2)
ꢀ
M.p.: 144–146 °C; IR (KBr): m = 3,355, 1,684,
1,596 cm-1 1H NMR (500 MHz, CDCl3): d = 1.34 (s,
;
9H, t-Bu), 5.81 (brs, 1H, NH), 6.14 (s, 1H, CH), 6.97–7.63
(m, 13H, HAr), 8.09 (d, 1H, J = 7.5 Hz, HAr), 9.53 (brs,
1H, NHindole
)
ppm; 13C NMR (125 MHz, CDCl3):
d = 28.7, 51.7, 66.9, 104.4, 107.0, 107.3, 111.5, 120.1,
122.4, 124.7, 127.9, 128.5, 128.7, 129.1, 129.6, 129.8,
130.2, 131.4, 132.3, 133.3, 135.5, 139.5, 142.0, 162.3,
168.9 ppm; HR-ESI–MS: calc. for C27H27IN3O2
([M ? 1]?) 552.11425, found 552.11430.
Experimental
N-[(tert-Butylcarbamoyl)(phenyl)methyl]-N-benzyl-1H-
All reagents obtained from commercial suppliers (Aldrich,
Fluka, Sigma, Merck) were of analytical grade and were
used without further purification. All the melting points
were determined on an Electrothermal 9100 apparatus. All
the IR spectra were recorded by using a Thermo Nicolet IR
100 FT-IR spectrometer. All 1H NMR and 13C NMR
spectra were recorded in CDCl3 and DMSO-d6 on a Bruker
DRX-500 spectrometer at 500 MHz for 1H NMR and
125 MHz for 13C NMR. High-resolution mass spectra were
measured on Mass ESI POS (ICR Apex-Qe instrument).
X-ray structural data were obtained by using Bruker APEX
II Quazar.
indole-2-carboxamide (5c, C28H29N3O2)
ꢀ
M.p.: 178–180 °C; IR (KBr): m = 3,312, 1,685,
1,587 cm-1 1H NMR (500 MHz, DMSO-d6): d = 1.21
;
(s, 9H, t-Bu), 4.86 (brs, 1H, CHBenzyl), 5.17 (brs, 1H,
CHBenzyl), 6.17 (s, 1H, CH), 6.58 (brs, 1H, NH), 7.05–7.93
(m, 15H, HAr), 11.72 (brs, 1H, NHindole) ppm; 13C NMR
(125 MHz, DMSO-d6): d = 28.5, 51.6, 66.0, 105.9, 111.7,
120.5, 122.3, 124,8, 126.9, 127.0, 127.8, 128.4, 128.5,
128.6, 128.8, 129.9, 134.6, 135.9, 137.3, 164.9, 168.6 ppm;
HR-ESI–MS: calc. for C28H30N3O2 ([M ? 1]?)
440.23355, found 440.23348.
N-[(tert-Butylcarbamoyl)(1H-indol-3-yl)methyl]-N-
(2-bromophenyl)-1H-indole-2-carboxamide
(5d, C29H27BrN4O2)
General procedure for synthesis of pseudopeptides
To a solution of aldehyde 1 (1 mmol) in 2 cm3 of MeOH
was added successively primary amine 2 (1 mmol), and the
mixture was stirred at room temperature for 1 h. Then acid
3 (1 mmol) was added, and stirring was continued for
15 min, followed by addition of isocyanide 4 (1 mmol).
After stirring for 24 h at room temperature and regular
control of the reaction progress by TLC, pure compound 5
was precipitated. The compound was filtered and washed
with MeOH.
M.p.: 218–220 °C; IR (KBr): mꢀ = 3,308, 1,675,
1,594 cm-1 1H NMR (500 MHz, CDCl3): d = 1.30 (s,
;
9H, t-Bu), 5.17 (brs, 1H, NH), 6.03 (s, 1H, CH), 6.97–7.62
(m, 12H, HAr), 7.66 (d, 1H, J = 7.2 Hz, HAr), 8.10 (d, 1H,
J = 7.8 Hz, HAr), 8.45 (brs, 1H, NHindole), 9.56 (brs, 1H,
NHindole) ppm; 13C NMR (125 MHz, CDCl3): d = 28.6,
51.5, 58.4, 103.2, 106.6, 107.3, 111.1, 111.5, 118.4, 119.4,
119.5, 121.0, 122.1, 124.6, 125.2, 127.0, 127.2, 128.6,
129.9, 131.9, 132.4, 133.3, 133.6, 135.5, 139.2, 162.4,
169.4 ppm; HR-ESI–MS: calc. for C29H28BrN4O2
([M ? 1]?) 543.13902, found 543.13899.
N-[(tert-Butylcarbamoyl)(phenyl)methyl]-N-
(2 bromophenyl)-1H-indole-2-carboxamide
(5a, C27H26BrN3O2)
X-ray structure analysis: colorless crystal (polyhedron),
dimensions 0.36 9 0.16 9 0.07 mm3, crystal system tri-
˚
clinic, space group P1ꢀ, Z = 2, a = 9.7637(2) A,
ꢀ
M.p.: 231–232 °C; IR (KBr): m = 3,324, 1,679,
1,601 cm-1
;
1H NMR (500 MHz, CDCl3): d = 1.34 (s,
9H, t-Bu), 5.90 (brs, 1H, NH), 6.17 (s, 1H, CH), 7.00–7.40
˚
˚
b = 11.7903(2) A, c = 12.8418(2) A, a = 70.116(1)°,
3
˚
(m, 13H, HAr), 8.09 (d, 1H, J = 7.5 Hz, HAr), 9.57 (brs,
ppm; 13C NMR (125 MHz, CDCl3):
b = 80.601(1)°, c = 89.209(1)°, V = 1,370.13(4) A ,
1H, NHindole
)
q = 1.395 g/cm3, T = 200(2) K, hmax = 32.03°, radiation
˚
d = 28.6, 51.7, 66.9, 106.7, 111.6, 120.2, 122.4, 124.7,
126.5, 127.9, 128.3, 128.4, 128.6, 129.3, 129.6, 130.3,
131.0, 132.1, 132.6, 133.2, 133.8, 135.5, 138.8, 162.4,
Mo Ka, k = 0.71073 A, 0.3° x-scans with CCD area
detector, covering the asymmetric unit in reciprocal space
with a mean redundancy of 3.89 and a completeness of
123

Indole-containing pseudopeptides
353
˚
CHBenzyl), 5.56 (brs, 1H, NH), 6.21 (s, 1H,
CH),7.14–7.32 (m, 13H, HAr), 7.98 (d, 1H, J = 8.1 Hz,
HAr), 9.54 (brs, 1H, NHindole) ppm; 13C NMR (125 MHz,
CDCl3): d = 24.6, 25.4, 32.6, 48.6, 50.7, 65.3, 110.3,
111.8, 120.8, 121.4, 123.0, 126.4, 126.5, 127.1, 127.2,
128.6, 128.8, 130.7, 134.0, 134.3, 135.9, 137.7, 168.7,
169.4 ppm; HR-ESI–MS: calc. for C30H31ClN3O2
([M ? 1]?) 500.20993, found 500.21000.
99.7 % to a resolution of 0.67 A, 37,106 reflections mea-
sured, 9,505 unique (R(int) = 0.0333), 7,774 observed
(I [ 2r(I)), intensities were corrected for Lorentz and
polarization effects, an empirical absorption correction was
applied using SADABS (Sheldrick) based on the Laue
symmetry of the reciprocal space, l = 1.54 mm-1
,
Tmin = 0.61, Tmax = 0.90, structure solved by direct
methods and refined against F2 with a full-matrix least-
squares algorithm using the SHELXTL (version 2008/4)
software package, 356 parameters refined, hydrogen atoms
were treated using appropriate riding models, except of
those at the nitrogen atoms, which were refined isotropi-
cally, goodness of fit 1.02 for observed reflections, final
residual values R1(F) = 0.035, wR(F2) = 0.086 for
observed reflections, residual electron density -0.25 to
N-[(tert-Butylcarbamoyl)(4-bromophenyl)methyl]-N-
benzyl-1H-indole-3-carboxamide (5h, C28H28BrN3O2)
ꢀ
M.p.: 132–134 °C; IR (KBr): m = 3,207, 1,669,
1,594 cm-1 1H NMR (500 MHz, CDCl3): d = 1.26 (s,
;
9H, t-Bu), 4.65 (d, 1H, J = 15.2 Hz, CHBenzyl), 4.88 (d,
1H, J = 15.2 Hz, CHBenzyl), 5.50 (brs, 1H, NH), 6.18 (s,
1H, CH), 7.11–7.39 (m, 13H, HAr), 7.97 (d, 1H,
J = 7.6 Hz, HAr), 9.54 (brs, 1H, NHindole) ppm; 13C
NMR (125 MHz, CDCl3): d = 28.5, 51.6, 65.8, 110.3,
111.8, 120.7, 121.4, 122.4, 123.0, 126.4, 126.5, 127.1,
127.2, 128.6, 130.9, 131.7, 134.6, 135.8, 137.7, 168.8,
169.5 ppm; HR-ESI–MS: calc. for C28H29BrN3O2
([M ? 1]?) 518.14377, found 518.14379.
0.57 eA-3. CCDC 857254 contains the supplementary
˚
crystallographic data for this article. These data can be
obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via http://www.ccdc.cam.ac.uk/data_
request/cif.
N-[(tert-Butylcarbamoyl)(4-chlorophenyl)methyl]-N-
benzyl-1H-indole-2-carboxamide (5e, C28H28ClN3O2)
N-[(Cyclohexylcarbamoyl)(4-bromophenyl)methyl]-N-
-1
ꢀ
M.p.: 204–206 °C; IR (KBr): m = 3,299, 1,653, 1,611 cm
;
benzyl-1H-indole-3-carboxamide (5i, C30H30BrN3O2)
ꢀ
1H NMR (500 MHz, CDCl3): d = 1.28 (s, 9H, t-Bu), 4.90 (d,
1H, J = 16.6 Hz, CHBenzyl), 5.31 (brs, 1H, CHBenzyl), 5.72
(brs, 1H, NH), 5.88 (s, 1H, CH), 6.80–7.41 (m, 13H, HAr),
7.54 (d, 1H, J = 8.5 Hz, HAr), 9.62 (brs, 1H, NHindole) ppm;
13C NMR (125 MHz, CDCl3): d = 28.5, 51.7, 65.1, 106.0,
111.7, 120.6, 122.4, 125.0, 126.9, 127.2, 127.8, 128.5, 128.6,
128.6, 131.2, 133.1, 134.5, 135.9, 137.1, 164.9, 168.2 ppm;
HR-ESI–MS: calc. for C28H29ClN3O2 ([M ? 1]?)
474.19428, found 474.19427.
-1
M.p.: 128–130 °C; IR (KBr): m = 3,240, 1,666, 1,603 cm
;
1H NMR (500 MHz, CDCl3): d = 1.00–1.81 (m, 10H,
Cyclohexyl), 3.68 (m, 1H, HCyclohexyl), 4.66 (d, 1H,
H
J = 16.5 Hz, CHBenzyl), 4.85 (d, 1H, J = 16.5 Hz, CHBen-
zyl), 5.52 (brs, 1H, NH), 6.22 (s, 1H, CH),7.14–7.40 (m, 13H,
HAr), 7.98 (d, 1H, J = 7.1 Hz, HAr), 9.43 (brs, 1H, NHindole
)
ppm; 13C NMR (125 MHz, CDCl3): d = 24.6, 25.4, 32.6,
48.6, 50.7, 65.4, 110.3, 111.8, 120.8, 121.4, 122.4, 123.0,
126.4, 126.5, 127.1, 127.2, 128.6, 131.0, 131.7, 134.5, 135.8,
137.7, 168.6, 169.4 ppm; HR-ESI–MS: calc. for
C30H31BrN3O2 ([M ? 1]?) 544.15942, found 544.15947.
N-[(tert-Butylcarbamoyl)(4-chlorophenyl)methyl]-N-
benzyl-1H-indole-3-carboxamide (5f, C28H28ClN3O2)
ꢀ
M.p.: 132–134 °C; IR (KBr): m = 3,208, 1,672,
N-[(tert-Butylcarbamoyl)(phenyl)methyl]-N-
(2-bromophenyl)-3-(1H-indol-3-yl)acetamide
(5j, C28H28BrN3O2)
1,597 cm-1 1H NMR (500 MHz, CDCl3): d = 1.26 (s,
;
9H, t-Bu), 4.65 (d, 1H, J = 16.2 Hz, CHBenzyl), 4.88 (d,
1H, J = 16.2 Hz, CHBenzyl), 5.52 (brs, 1H, NH), 6.16 (s,
1H, CH), 7.11–7.30 (m, 13H, HAr), 7.97 (d, 1H,
J = 7.7 Hz, HAr), 9.53 (brs, 1H, NHindole) ppm; 13C
NMR (125 MHz, CDCl3): d = 28.5, 51.6, 65.7, 110.3,
111.8, 120.7, 121.4, 123.0, 126.4, 126.5, 127.1, 127.2,
128.6, 128.7, 130.7, 134.1, 134.2, 135.8, 137.8, 168.9,
169.5 ppm; HR-ESI–MS: calc. for C28H29ClN3O2
([M ? 1]?) 474.19428, found 474.19427.
ꢀ
M.p.: 264–267 °C; IR (KBr): m = 3,275, 1,652,
1,548 cm-1 1H NMR (500 MHz, DMSO-d6): d = 1.21
;
Table 2 Lipophilicity measurement based on the shake flask method
and LC–MS/MS
Method
Shake flask method
MS conditions
Sciex API 3000, Triple
Quadrupole LC/MS/MS
Mass Spectrometer
N-[(Cyclohexylcarbamoyl)(4-chlorophenyl)methyl]-N-
Stock concentration
Solubility
10 mM
100 % DMSO
10 lM
benzyl-1H-indole-3-carboxamide (5g, C30H30ClN3O2)
ꢀ
M.p.: 129–132 °C; IR (KBr): m = 3,246, 1,668,
Test concentration
Final DMSO concentration in assay
Incubation time
1
1,605 cm-1; H NMR (500 MHz, CDCl3): d = 1.01–1.80
(m, 10H, HCyclohexyl), 3.72 (m, 1H, HCyclohexyl), 4.65 (d, 1H,
0.1 %
1 h
J = 16.5 Hz, CHBenzyl), 4.89 (d, 1H, J = 16.5 Hz,
123

354
N. S. Alavijeh et al.
Table 3 Experimentally obtained measures of lipophilicity
Entry
Phases
Peak area
EXP1
Mean
SEM
CV/%
EXP2
EXP3
5a
n-Octanol
PBS
631,665.00
518,887.00
65,328.00
893,469.00
697,552.00
69,976.00
800,572.00
595,855.00
53,082.00
11,262.00
425,684.00
16,232.00
38,104.00
6,622.00
775,235.33
604,098.00
62,795.33
13,399.33
434,432.67
15,175.33
50,116.00
6,356.67
76,630.70
51,740.56
5,038.60
1,194.94
47,732.95
882.80
17.12
14.83
13.90
15.45
19.03
10.08
22.39
19.41
15.89
19.96
5.03
5b
n-Octanol
PBS
15,394.00
13,542.00
5c
n-Octanol
PBS
356,479.00
13,422.00
521,135.00
15,872.00
5d
n-Octanol
PBS
60,324.00
51,920.00
6,477.47
712.21
5,012.00
7,436.00
5e
n-Octanol
PBS
234,794.00
74,336.00
234,630.00
86,304.00
175,538.00
57,426.00
9,646,352.00
13,652.00
97,280.00
23,294.00
10,021,900.0
26,432.00
8,167,352.00
13,120.00
113,448.00
10,194.00
130,200.00
15,414.00
174,876.00
25,950.00
204,734.00
67,558.00
145,409.00
1,634.00
214,987.33
72,688.67
9,989,850.67
16,469.33
132,076.67
30,590.67
10,513,860.0
30,325.33
8,388,661.33
16,833.33
137,342.00
13,594.00
159,539.33
19,157.33
205,630.00
34,412.67
260,933.33
84,646.67
140,173.33
1,571.67
19,724.72
8,376.95
289,852.01
1,694.10
17,495.73
3,957.27
351,331.65
4,096.99
217,367.32
2,003.94
12,276.98
1,789.15
14,732.86
2,074.35
15,762.69
4,623.12
28,153.25
8,923.41
9,169.20
34.72
5f
n-Octanol
PBS
10,566,012.00
19,508.00
9,757,188.00
16,248.00
17.82
22.94
22.41
5.79
5g
n-Octanol
PBS
152,668.00
36,894.00
146,282.00
31,584.00
5h
n-Octanol
PBS
11,194,332.00
38,516.00
10,325,348.0
26,028.00
23.40
4.49
5i
n-Octanol
PBS
8,823,372.00
19,996.00
8,175,260.00
17,384.00
20.62
15.48
2,280
15.99
18.75
13.28
23.27
18.69
18.26
11.33
3.83
5j
n-Octanol
PBS
154,186.00
16,260.00
144,392.00
14,328.00
5k
n-Octanol
PBS
171,848.00
22,578.00
176,570.00
19,480.00
5l
n-Octanol
PBS
215,008.00
35,418.00
227,008.00
41,870.00
5m
5n
n-Octanol
PBS
292,040.00
97,648.00
286,026.00
88,734.00
n-Octanol
PBS
122,335.00
1,567.00
152,776.00
1,514.00
5o
n-Octanol
PBS
134,342.00
28,944.00
135,600.00
31,960.00
108,594.00
24,168.00
939,096.00
8,960.00
126,178.67
28,357.33
946,380.67
10,796.67
80,391.00
1,960,428.67
8,000,812.00
1,680,486.00
12,656,729.3
699,693.33
153,215.33
8,036,384.00
8,799.83
2,268.40
100,767.86
1,347.08
5,334.92
23,865.27
406,415.98
33,233.80
481,316.04
38,447.29
9,087.25
104,217.19
12.08
13.86
18.44
21.61
11.49
2.11
Ref1
Ref2
Ref3
Ref4
Ref5
n-Octanol
PBS
775,602.00
13,422.00
1,124,444.00
10,008.00
n-Octanol
PBS
85,062.00
86,384.00
69,748.00
2,002,448.00
7,407,570.00
1,731,424.00
12,187,444.0
748,274.00
153,544.00
7,853,154.00
1,919,812.00
7,816,216.00
1,691,996.00
12,163,482.00
623,784.00
137,314.00
8,214,044.00
1,959,026.00
8,778,650.00
1,618,038.00
13,619,262.0
727,022.00
168,788.00
8,041,954.00
n-Octanol
PBS
8.80
3.43
n-Octanol
PBS
6.59
9.52
n-Octanol
PBS
10.27
2.25
Data shown are mean standard error of the mean (SEM), with coefficient of variation (CV)
Ref1, ketoconazole; Ref2, tetracycline; Ref3, propranolol; Ref4, verapamil; Ref5, atenolol
(s, 9H, t-Bu), 3.32 (d, 1H, J = 17.5 Hz, CH2), 3.37 (d, 1H,
J = 17.5 Hz, CH2), 5.97 (s, 1H, CH), 6.89 (brs, 1H, NH),
7.06–7.70 (m, 13H, HAr), 8.03 (d, 1H, J = 6.0 Hz, HAr),
10.81 (brs, 1H, NHindole) ppm; 13C NMR (125 MHz,
DMSO-d6): d = 28.3, 31.7, 50.2, 64.5, 108.0, 111.1, 118.1,
118.7, 120.7, 123.8, 125.6, 127.2, 127.3, 127.8, 127.9,
123

Indole-containing pseudopeptides
355
129.7, 130.5, 132.4, 133.7, 133.8, 136.0, 138.8, 169.3,
170.0 ppm; HR-ESI–MS: calc. for C28H29BrN3O2
([M ? 1]?) 518.14377, found 518.14373.
CH2), 5.79 (brs, 1H, NH), 5.98 (s, 1H, CH), 6.91–7.37 (m,
13H, HAr), 7.88 (d, 1H, J = 7.6 Hz, HAr), 8.09 (brs, 1H,
NHindole) ppm; 13C NMR (125 MHz, CDCl3): d = 20.9,
28.7, 35.9, 51.6, 65.6, 111.0, 115.3, 118.7, 119.0, 121.6,
121.7, 126.0, 127.3, 127.8, 128.1, 128.4, 129.6, 129.8,
131.6, 130.9, 132.8, 133.0, 133.4, 136.2, 138.6, 169.4,
173.2 ppm; HR-ESI–MS: calc. for C29H31BrN3O2
([M ? 1]?) 532.15967, found 532.15962.
N-[(tert-Butylcarbamoyl)(phenyl)methyl]-3-(1H-indol-3-
yl)-N-(2-iodophenyl)acetamide (5k, C28H28IN3O2)
ꢀ
M.p.: 268–270 °C; IR (KBr): m = 3,269, 1,651,
1,546 cm-1 1H NMR (500 MHz, DMSO-d6): d = 1.20
;
(s, 9H, t-Bu), 3.28 (d, 1H, J = 17.5 Hz, CH2), 3.34 (d, 1H,
J = 17.5 Hz, CH2), 5.95 (s, 1H, CH), 6.85–7.68 (m, 13H,
HAr), 7.13 (brs, 1H, NH), 8.02 (d, 1H, J = 7.9 Hz, HAr),
10.81 (brs, 1H, NHindole) ppm; 13C NMR (125 MHz,
DMSO-d6): d = 28.3, 32.4, 50.1, 64.6, 105.1, 108.1, 111.1,
118.1, 118.9, 120.7, 123.9, 127.1, 127.3, 127.8, 128.7,
129.5, 130.9, 133.0, 133.6, 136.0, 138.7, 142.2, 169.4,
169.9 ppm; HR-ESI–MS: calc. for C28H29IN3O2
([M ? 1]?) 566.12990, found 566.12992.
N-[(tert-Butylcarbamoyl)(phenyl)methyl]-3-(1H-indol-3-
yl)-N-(2-iodophenyl)propanamide (5o, C29H30IN3O2)
ꢀ
M.p.: 140–142 °C; IR (KBr): m = 3,340, 1,643,
1,534 cm-1
;
1H NMR (500 MHz, CDCl3): d = 1.36 (s,
9H, t-Bu), 2.35–2.48 (m, 2H, CH2), 3.06–3.15 (m, 2H,
CH2), 5.74 (br s, 1H, NH), 5.98 (s, 1H, CH), 6.79–7.54 (m,
13H, HAr), 7.90 (d, 1H, J = 7.7 Hz, HAr), 8.04 (br s, 1H,
NHindole) ppm; 13C NMR (125 MHz, CDCl3): d = 20.9,
28.7, 36.5, 51.6, 65.7, 104.2, 111.0, 115.4, 118.8, 119.1,
121.7, 121.7, 127.3, 127.7, 128.4, 129.0, 129.5, 130.1,
131.4, 132.7, 132.9, 136.2, 139.2, 141.9, 169.4,
173.1 ppm; HR-ESI–MS: calc. for C29H31IN3O2
([M ? 1]?) 580.14555, found 580.14557.
N-[(tert-Butylcarbamoyl)(4-chlorophenyl)methyl]-N-
benzyl-3-(1H-indol-3-yl)acetamide (5l, C29H30ClN3O2)
ꢀ
M.p.: 193–195 °C; IR (KBr): m = 3,210, 1,654,
1,521 cm-1 1H NMR (500 MHz, CDCl3): d = 1.29 (s,
;
9H, t-Bu), 3.79 (d, 1H, J = 15.1 Hz, CH2), 3.84 (d, 1H,
J = 16.7 Hz, CH2), 4.56 (d, 1H, J = 17.1 Hz, CHBenzyl),
4.85 (d, 1H, J = 17.1 Hz, CHBenzyl), 5.81 (brs, 1H, NH),
5.91 (s, 1H, CH), 6.96–7.33 (m, 13H, HAr), 7.46 (d, 1H,
J = 7.7 Hz, HAr), 8.46 (brs, 1H, NHindole) ppm; 13C NMR
(125 MHz, CDCl3): d = 28.5, 32.1, 50.5, 51.7, 62.7,
108.6, 111.3, 118.8, 119.5, 122.1, 122.9, 123.8, 126.1,
127.1, 128.5, 128.7, 131.0, 133.7, 134.3, 136.3, 137.5,
168.7, 173.6 ppm; HR-ESI–MS: calc. for C29H31ClN3O2
([M ? 1]?) 488.20993, found 488.20992.
Table 4 Experimentally obtained measures of lipophilicity based on
log D (pH 7.4)
Entry
Log D
EXP1
SEM
pH 7.4
EXP2
EXP3
Mean
5a
0.09
0.63
1.42
1.08
0.50
2.73
0.62
2.46
2.64
0.98
0.88
0.78
0.48
1.89
0.67
1.76
-1.35
0.66
1.29
-1.78
0.11
0.71
1.52
0.84
0.43
2.78
0.67
2.60
2.67
1.00
0.96
0.73
0.51
2.00
0.63
2.05
-1.36
0.73
1.27
-1.68
0.13
0.67
1.42
0.76
0.49
2.85
0.62
2.58
2.79
1.05
0.93
0.83
0.48
1.95
0.65
2.02
-1.46
0.63
1.21
-1.71
0.11
0.67
1.45
0.89
0.47
2.79
0.63
2.55
2.70
1.01
0.92
0.78
0.49
1.95
0.65
1.94
-1.39
0.68
1.26
-1.72
0.01
0.02
0.03
0.10
0.02
0.03
0.02
0.04
0.05
0.02
0.02
0.03
0.01
0.03
0.01
0.09
0.03
0.03
0.02
0.03
5b
5c
N-[(Cyclohexylcarbamoyl)(4-chlorophenyl)methyl]-N-
5d
benzyl-3-(1H-indol-3-yl)acetamide (5m, C31H32ClN3O2)
ꢀ
5e
M.p.: 170–172 °C; IR (KBr): m = 3,248, 1,656,
1
1,623 cm-1; H NMR (500 MHz, CDCl3): d = 0.95–1.82
5f
(m, 10H, HCyclohexyl), 3.76 (m, 1H, HCyclohexyl), 3.80 (d, 1H,
J = 18.0 Hz, CH2), 3.83 (d, 1H, J = 16.5 Hz, CH2), 4.54
(d, 1H, J = 17.6 Hz, CHBenzyl), 4.83 (d, 1H, J = 17.6 Hz,
CHBenzyl), 5.84 (br s, 1H, NH), 5.93 (s, 1H, CH), 6.89–7.34
(m, 13H, HAr), 7.46 (d, 1H, J = 7.5 Hz, HAr), 8.43 (br s,
5g
5h
5i
5j
5k
1H, NHindole
)
ppm; 13C NMR (125 MHz, CDCl3):
5l
d = 24.7, 25.4, 32.1, 32.6, 48.7, 50.7, 62.5, 108.6, 111.3,
118.7, 119.6, 122.2, 123.0, 126.2, 127.2, 128.5, 128.7,
131.1, 133.6, 134.4, 136.3, 137.3, 168.4, 173.5 ppm; HR-
MS (ESI) calc. for C31H33ClN3O2 ([M ? 1]?) 514.22558,
found 514.22561.
5m
5n
5o
Ref1
Ref2
Ref3
Ref4
Ref5
N-[(tert-Butylcarbamoyl)(phenyl)methyl]-N-
(2-bromophenyl)-3-(1H-indol-3-yl)propanamide
(5n, C29H30BrN3O2)
M.p.: 190–191 °C; IR (KBr): mꢀ = 3,328, 1,636,
Data shown are mean standard error of the mean (SEM)
1,542 cm-1 1H NMR (500 MHz, CDCl3): d = 1.36 (s,
;
9H, t-Bu), 2.39–2.45 (m, 2H, CH2), 3.07–3.13 (m, 2H,
Ref1, ketoconazole; Ref2, tetracycline; Ref3, propranolol; Ref4,
verapamil; Ref5, atenolol
123

356
N. S. Alavijeh et al.
3. Katsman A, Umezawa K, Bonavida B (2009) Curr Pharm Des
15:792
Lipophilicity
4. Aboul-Enein HY, Kruk I, Lichszteld K, Michalska T, Kiadna A,
Marczynski S, Olgen S (2004) Luminescence 19:1
5. Bellemin R, Decerprit J, Festal D (1996) Eur J Med Chem 31:123
6. Battaglia S, Boldrini E, Settimo FD, Dondio G, Motta CL, Marini
AM, Primofiore G (1999) Eur J Med Chem 34:93
7. Carbonnelle D, Duflos M, Marchand P, Chauvet C, Petit JY, Lang
F (2009) J Pharmacal Exper Ther 331:710
8. Grumel V, Merour JY, Lesur B, Giboulot T, Frydman A, Guil-
laumet G (2002) Eur J Med Chem 37:45
9. Kitano M, Kojima A, Nakano K, Miyagishi A, Noguchi T, Oh-
ashi N (1999) Chem Pharm Bull 47:1538
10. Sewald N, Jakubke HD (eds) (2002) Peptides: chemistry and
biology. Wiley-VCH, Weinheim
11. Sengupta A, Mahalakshmi R, Shamala N, Balaram P (2005) J
Pept Res 65:113
12. Duan G, Smith VH, Weaver DF (1999) Chem Phys Lett 310:323
13. Cudic P, Stawikowski M (2007) Mini Rev Org Chem 4:268
14. Nielsen PE (ed) (2004) Pseudo-peptides in drug discovery.
Wiley-VCH, Weinheim
15. Slobbe P, Ruijter E, Orru RVA (2012) Med Chem Commun
3:1189
The most common method of log D determination is the
shake-flask method. This method involves dissolving the
compound in a known volume of octanol (hydrophobic)
and water or PBS (hydrophilic), then measuring the con-
centration of the compound in each phase. The properties
of octanol (i.e., hydrophobic) resemble those of lipid
bilayer membranes on cells. Hydrophobic drugs (high
partition coefficients) are preferentially distributed to
hydrophobic compartments of cells such as the lipid
bilayers. Hydrophilic drugs (low partition coefficients) are
found in hydrophilic compartments such as blood serum.
Compound levels in both layers are quantified using a LC-
MS/MS. All measurements were repeated at least three
times. Results were expressed as log D (pH 7.4) from the
equation log D (pH 7.4) = log10 (peak area for n-octanol
sample/peak area for buffer sample).
Table 2 shows the mass spectrometry (MS) instrument
conditions used for the detection of these compounds. It is
an analytical tool used for measuring the molecular mass of
a sample in drug discovery, combinatorial chemistry,
pharmacokinetics, and drug metabolism studies. Table 2
also shows the experimental conditions of the log D assay,
such as drug concentration and incubation time. Table 3
shows the raw data from the mass spectrometry instrument;
the results from both compartments are shown, from the
octanol (hydrophobic) and PBS (hydrophilic) parts.
Table 4 shows the experimentally obtained measures of
lipophilicity based log D (pH 7.4), derived from the
equation log D (pH 7.4) = log10 (peak area for n-octanol
sample/peak area for buffer sample).
¨
16. Domling A, Wang W, Wang K (2012) Chem Rev 112:3083
17. Akritopoulou-Zanze I (2008) Curr Opin Chem Biol 12:324
18. Sapi J, Laronze JY (2004) Arkivoc vii:208
19. Balalaie S, Bigdeli MA, Sheikhhosseini E, Habibi A, Piri H,
Naderi M (2012) Helv Chim Acta 95:528
20. Haghighatnia Y, Balalaie S, Bijanzadeh HR (2012) Helv Chim
Acta 95:818
21. Balalaie S, Motaghedi H, Bararjanian M, Tahmassebi D, Bijan-
zadeh HR (2011) Tetrahedron 67:9134
22. Balalaie S, Bararjanian M, Rominger F, Hosseinzadeh S, Bijan-
zadeh HR, Wolf E (2011) Tetrahedron 67:7294
23. Bararjanian M, Hosseinzadeh S, Balalaie S, Bijanzadeh HR
(2011) Tetrahedron 67:2644
24. Bararjanian M, Balalaie S, Rominger F, Movassagh B, Bijan-
zadeh HR (2010) J Org Chem 75:2806
¨
25. Domling A (2006) Chem Rev 106:17
¨
26. Domling A, Ugi I (2000) Angew Chem Int Ed 39:3168
27. Hansch C (1981) Drug Dev Res 1:267
28. Testa B, Van de Waterbeemd H, Folkers G, Guy R (eds) (2001)
Pharmacokinetic optimization in drug research. Wiley-VCH,
Weinheim
Acknowledgments We gratefully thank the Iran National Science
Foundation (INSF) for the financial support.
29. Rodgers SL, Davis AM, Tomkinson NP, Van de Waterbeemd H
(2011) Mol Inf 30:256
30. Saha P, Kim KJ, Yamahara H, Crandall ED, Lee VHL (1994) J
Control Release 32:191
31. Di L, Kerns EH (2003) Curr Opin Chem Biol 7:402
References
1. Ahmad A, Sakr WA, Rahman KM (2011) Cancers 3:2955
2. Lodyga-Chruscinska E, Turek M (2009) Pharma Chem 8:6
123