Synthesis of novel, peptidic kinase inhibitors with cytostatic/cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2014.01.005

The utility of a novel, chemoenzymatic procedure for the stereocontrolled synthesis of small peptides is presented in the preparation and structure optimisation of dipeptides with cytostatic/cytotoxic activity. The method uses Passerini multicomponent rea

Full text of DOI:10.1016/j.bmc.2014.01.005

Accepted Manuscript
Synthesis of Novel, Peptidic Kinase Inhibitors with Cytostatic/Cytotoxic Ac-
tivity
Wiktor Szymanski, Magdalena Zwolinska, Szymon Klossowski, Izabela
Młynarczuk-Biały, Łukasz Biały, Tadeusz Issat, Jacek Malejczyk, Ryszard
Ostaszewski
PII:
S0968-0896(14)00023-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.01.005
BMC 11336
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 May 2013
20 December 2013
3 January 2014
Please cite this article as: Szymanski, W., Zwolinska, M., Klossowski, S., Młynarczuk-Biały, I., Biały, Ł., Issat, T.,
Malejczyk, J., Ostaszewski, R., Synthesis of Novel, Peptidic Kinase Inhibitors with Cytostatic/Cytotoxic Activity,
Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2014.01.005
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical abstract:
Synthesis of Novel, Peptidic Kinase Inhibitors with Cytostatic/Cytotoxic
Activity
Wiktor Szymanski,a Magdalena Zwolinska,a Szymon Klossowskid, Izabela Młynarczuk-
Biały,b Łukasz Białyb, Tadeusz Issatc , Jacek Malejczyk b and Ryszard Ostaszewski*d
a Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664
Warsaw, Poland
b Department of Histology and Embryology, the Medical University of Warsaw,5
Chalubinskiego Str., 02-004, Warsaw, Poland
c Department of Immunology, the Medical university of Warsaw 5 Chalubinskiego Str.,
02-004, Warsaw, Poland
d Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224
Warsaw, Poland Fax: +48 22 632 66 81; Tel: +48 22 343 21 20; E-mail:
rysza@icho.edu.pl
Abstract: The utility of a novel, chemoenzymatic procedure for the stereocontrolled
synthesis of small peptides is presented in the preparation and structure optimisation of
dipeptides with cytostatic/cytotoxic activity. The method uses Passerini multicomponent
reaction for the preparation of racemic scaffold which is then enantioselectivelly
hydrolysed by hydrolytic enzymes. Products of these transformations are further
functionalised towards title compounds. Both activity and selectivity towards tumor cells
is optimised. Final compound is shown to be an inhibitor of the protein kinase signaling
pathway.
1

Keywords: peptidomimetics, biotransformations, cytostatic/cytotoxic effect, kinase
inhibitors
1. Introduction
The promising use of small peptides as therapeutics suffers seriously from the problems
with pharmacokinetics of these compounds. These problems include proteolytic
instability and high polarity, which prevents small peptides from crossing cell membranes.
The synthesis of peptidomimetics aims at the solution of these problems. The changes,
which are usually introduced into the structure of the peptide are: N-alkylation of peptide
bond,1 the incorporation of non-coded amino acids2 and the introduction of special
functional groups at the termini of the peptide.3
A novel method for the preparation of peptidomimetics of structure 7 has been recently
developed in our group (Scheme 1).4 It uses Passerini multicomponent reaction for the
preparation of racemic scaffolds rac-4 which is then enantioselectivelly hydrolysed by
hydrolytic enzymes to the enantiomerically enriched alcohols 5, leaving the enriched
esters 4. A method for the conversion of compounds 4 and 5 into each other, with the
inversion of configuration, has also been established,5 allowing a more efficient synthesis
of a chosen enantiomer. Alcohols 5 are then transformed into amines 6 (by an SN2 type
reaction of sodium azide with methanesulfonic ester of 5, followed by the reduction of
the azide), which are subsequently coupled with N-protected amino acids to yield the title
compounds 7 (R3 = H). An alternative route leads to N-methylamines 6 (R3 = Me), by
reacting methanesulfonic ester of 5 with aqueous methylamine. These compounds are
further coupled with N-protected amino acids to yield title compounds 7 (R3 = Me),
derivatives of N-alkylated amino acids.
Mitogen-Activated Protein Kinase (MAPK) pathway is essential for induction of cell
proliferation and may be hyperactivated in various tumor cells 6,7. This pathway consists
of small, guanine nucleoside-binding proteins, RAS, that regulate multiple signal
transduction pathways, including the activation of downstream RAF-MEK1/2-ERK1/2
2

protein kinase cascade7,8. Inhibition of this cascade is known to block activation and
subsequent cell proliferation, and at least two inhibitors of the pathway were shown to
have promising antitumor effects in vitro and in vivo.9,10
In this paper we describe our studies on the use of above-described methodology for the
preparation and optimization of a group of dipeptides with selective effect on MAPK
kinase pathway that would exert cytostatic/cytotoxic effects towards selected murine
tumor cell line displaying Ras overactivation (RAS-3T3 cells). This work was inspired by
the observation, that for protein kinase inhibitors, the amide bond is essential for maintain
the activity,11 which is consistent with the natural substrate scope of the kinases.
2. Results and discussion
2.1 The optimisation of R1 fragment derived from isocyanide
An analysis of the ideal structure of MAPK pathway inhibitor, reported by Yingchun et
all,11 reveals that the peptide bond should have two hydrophobic substituents.11 Therefore,
in this part of the study, the R2 substituent was fixed as n-heptyl chain, and a set of
hydrophobic groups was tested as R1.(Scheme 2)
Racemic compounds 7, bearing a glycine fragment (R4 = H) and no N-modification (R3 =
H) were synthesized (Scheme 2) according to the previously established procedures.1
Isocyanides, the precursors of R1 fragment were either commercially available or were
synthesized by the dehydration of corresponding N-formylamines by POCl3/Et3N. Yields
of key transformations are given in Table 1, entries a-e. Racemic α-hydroxyamides 5
were synthesized in good overall yields, as both the Passerini reaction and the ester
hydrolysis step gave good results. The yield of Passerni reaction was slightly lowered
only in the case of cyclohexyl isocyanide (1a), probably due to the steric hindrance on the
α carbon. The three-step transformation of hydroxy group into an amino group was
performed with overall yields exceeding 75% and the coupling steps gave products with
excellent yields (>85%).
3

Results of the bioactivity study for the compounds 7a-e are presented in Table 2. All the
compounds displayed cytotoxic/cytostatic activity against both tumor (RAS-3T3) and
non-tumor (NIH-3T3) cell lines, however with different selectivity. Compounds 7d and
7e showed no selectivity as their IC50 was similar for both cell lines. Compounds 7a-c
were more selective towards RAS-3T3 cell line, which might suggests possible
interaction with the MAP kinase pathway. The best result was obtained for compound 7c,
possessing the phenethyl moiety in the optimized fragment.
2.2 The optimisation of R2 fragment derived from the aldehyde
The same methodology was used for the synthesis of a series of amides 7f-k (Table 1),
bearing a glycine fragment (R4 = H) and no N-modification (R3 = H). R1 fragment was set
as p-methoxybenzyl, due to the ease of synthesis and low volatility of respective
isocyanide.
All the compounds were obtained in good yields (Table 1), and only the synthesis of a
compound 7i with cyclohexyl group proved to be problematic, as the SN2 reaction with
sodium azide is very sensitive to the bulkiness of R2 substituent and gave product in only
15% yield.
Results of the bioactivity study for the compounds 7d and 7f-k (which all have the same
R1 fragment) are presented in Table 3. Out of all the studied structures, only those with
aliphatic R2 groups showed any cytostatic/cytotoxic effect. Significant selectivity was
reached only in compound 7k indicating that n-nonyl group is the best out of the studied
fragments.
2.3 The influence of N-methylation
The influence of the N-methylation of peptide bond (R3, Scheme 1) was also investigated.
N-methylated analog of 7k was prepared (Scheme 3) by reaction of respective
methansulfonic ester with aqueous methylamine. Thus obtained α-methylaminoamide
4

was coupled with N-protected glycine to give compound 7l. IC50 values for compound 7l
were 30 µM both for NIH-3T3 and RAS-3T3 cell lines. This indicates, that the studied
modification decreases the activity and selectivity and should be avoided in further study.
2.4 The optimisation of the amino acid-derived fragment (R4)
The influence of the type of N-terminal amino acid (R4, Scheme 1) was studied on four
different small peptides containing: glycine (7k), L-alanine (7m), ), L-leucine (7n) and L-
phenylalanine (7o). Synthesis of these compounds was achieved by the coupling of
respective amino acid to the α-aminoamide 6k.
Results of the bioactivity study for the compounds 7k and 7m-o are presented in Table 4.
They indicate, that the introduction of R4 substituent results in higher selectivity toward
tumor cell lines. However, this introduction resulted in lowering the activity of the
compounds.
2.5 Determination of the active enantiomer
Enantiomerically enriched α-hydroxyamides 5k, precursors of 7k, were obtained in
Lipase from porcine pancreas (PPL)-catalyzed hydrolysis of racemic ester 4k (Scheme 5).
Further modifications (according to Scheme 2) enabled the synthesis of enantiomerically
enriched compounds (R)-7k and (S)-7k. Absolute configuration of these compounds was
assigned using CD spectroscopy (see supporting information for further details).
The effect of these compounds on the activity of MAP kinase cascade was evaluated by
means of in vitro MEK/ERK1 cascade assay using myelin basic protein (MBP) as a
substrate. As shown in Figure 1, compound 7k reduced, in a dose-dependent manner, the
level of BMP phosphorylation, thus suggesting that it inhibited MEK/ERK1 activity. The
inhibitory effect of compound (R)-7k was less than that of compound 7k. On the other
hand, compound (S)-7k was more active than 7k alone in reduction of MEK/ERK1
cascade activity suggesting that this form is more potent in kinase activity inhibition. The
5

difference was, however, not strongly pronounced, and therefore, compound7k in
racemic form was used for further experiments.
2.6 Inhibitory properties of the optimised compound
Basing on the gathered information, an optimised structure 7p was deduced (Scheme 4),
which possesses the best of the studied R1-R4 substituents. The results of the synthesis of
racemic compound 7p (according to Scheme 2) are collected in Table 1.
The results of evaluation of MEK/ERK1 kinase activity (Figure 1) strongly implied that
compound 7k inhibited either MEK or ERK1 or both. Thus, for characterization of
inhibitory activity of optimized compound 7p were performed in vitro experiments to
identify which MAP kinase is a potential target for the studied compound.
The effect of racemic compound 7p on MEK-mediated ERK phosphorylation was tested
on RAS-3T3 cells by means of Western-blot and specific quantitative Enzyme-Linked
Immunosorbent Assay (ELISA). U0216, a commercial inhibitor of MEK12 served as
control. Western-blot analysis (Figure 2A) has revealed that treatment of RAS-3T3 cells
with compound 7p almost completely inhibited phosphorylation of ERK1/2 and this
effect was comparable to the effect of U0216. Neither tested compound affected the
level of unphosphorylated ERK. Almost complete (90%) reduction of the pERK1/2 level
by treatment with compound 7p was also revealed by specific ELISA (Figure 2B).
Similar effect was also seen with U0126. These results clearly indicate that the
compound 7p is able to enter the cytoplasm of living cells where it affects ERK
phosphorylation, possibly by inhibition of MEK activity.
These results, however, do not exclude the possibility that compound 7p may also inhibit
activity of activated pERK1/2. Thus, we performed in vitro experiments with purified
activated ERK1 and MBP, as substrate. As shown in Figure 3A, the compound 7p
inhibited ERK1 activity in a dose-dependent manner. To reveal whether this compound
inhibitory activity was related to blocking of substrate binding site of EKR1 we
6

performed experiments with addition of increasing amounts of the substrate (MBP) as a
putative competitor. As shown in Figure 3B, the compound 7p-mediated ERK1 inhibition
was almost completely reversed by the maximal amount of MBP. This strongly suggests
that compound 7p is also a substrate-competitive inhibitor ofERK1.
Finally, we evaluated the activity of described 7p on other kinases. We performed the
activity test on thirteen enzymes: AurA, ALK, IGF1R, PIM1, FLT3, FGFR2, JAK2,
JAK3, BTK, TNIK, KDR, PDGFR-β, EGFR. (Figure 4). Compound 7p was tested with
U0126 as a reference at 10 µM concentration. Remarkably, we did not observed the
significant inhibition of studied kinases at this concentration. This indicates that, in
studied kinome, the obtained inhibitor is selective against ERK1.
3. Conclusion
We have presented the utility of a methodology, established in our group, for the
synthesis of nonnatural dipetide analogues. It enables a convenient introduction of
various substituents and peptide bond modifications, also allowing the stereocontrolled
synthesis of title compounds.
The cytostatix/cytotoxic effect of the synthesised compounds was evaluated on tumor cell
line (RAS-3T3) displaying overactivation of the MAP kinase RAS/MEK/ERK pathway
in comparison to the parental non-tumorigenic cells. This enabled selection of
compounds with the highest specificity against tumor cells. Final optimized compound
was found to be an inhibitor of both MEK and ERK protein kinases being key elements
of MAP kinase cascade pathway responsible for proliferation of tumor cells.
4. Experimental
4.1 Materials and Methods
Analytics:
7

Optical rotations were measured with a JASCO DIP-360 polarimeter. NMR spectra were
measured with a Varian 200 GEMINI and Varian 400 GEMINI spectrometer, with TMS
used as internal standard. TLCs were performed with silica gel 60 (230–400 mesh, Merck)
and silica gel 60 PF254 (Merck), respectively. MS spectra were recorded on an API - 365
(SCIEX) apparatus.
Reagents:
U0126 (Promega Madison, WI ) was maintained according to manufacturers instructions.
All synthesized substances were dissolved in DMSO as 10 mM stocks and stored at –
200C.
Cell lines:
Experiments were performed on murine NIH-3T3 fibroblasts (ATCC), stably transfected
with Ha-Ras oncogene (RAS-3T3 cells).14 Parental NIH-3T3 cells servedas control. The
cells were routinely cultured in DMEM supplemented with 2 mM 2-mercapthomethanol,
10% fetal bovine serum (FBS) and 1% antibiotic-antymycotic solution (all from Gibco)
at 37oC in humified atmosphere of 5% CO2 in air.
Growth retardation/cytotoxicity assay:
The cytostatic/cytotoxic effect of the studied compounds on RAS-3T3 and NIH-3T3 cells
was evaluated by crystal violet assay, as described in detail previously.14
Western-blot analysis of ERK1/2:
For examination of phosphorylation status of ERK1/2 RAS-3T3, cells were starved
overnight in the medium containing 2% FBS. Then the medium was replaced with a new
one containing 10% FBS and different concentrations of the tested compounds.
Following 4 hrs of incubation, 4 x 106 cells were lysed in lysis buffer (0.1% Triton X-
100, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 100 mM NaCl, supplemented with 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, 2 µg/ml leupeptin and 2 µg/mL
apoprotinin). Next, cell lysates were cleared by centrifugation (15 minutes, 14000 rpm at
4°C). Protein concentrations were determined by colorimetric bicinchoninic acid method
8

(Pierce). Lysate proteins (50 µg) were separated on 10% SDS-PAGE and transferred to
nitrocellulose membranes. Equal loading was confirmed by staining with 1% Ponceau
Red. Blots were blocked in 10% horse serum/5% (w/v) non-fat dry milk/0.4% Tween 20
in PBS for 1h and then labeled with rabbit polyclonal anti pERK antibody and goat anti
total ERK antibody (Santa Cruz, CA, USA) diluted in 2% dry milk/0.1% Tween 20 in
PBS. The immunoblots were visualized with horseradish peroxidase-conjugated
secondary antibodies (Jackson Immunoresearch, West Grove, PA) and enhanced by
cheminluminescence (Roche, Indianapolis, Indiana).
Enzyme-linked immunosorbent assay (ELISA) of pERK1/2:
Phosphorylated ERK1/2 (pERK1/2) was evaluated using commercial DuoSet ELISA
specific for pERK1/2 (R&D Systems, Abingdon, UK). For this purpose the cells were
pre-starved and treated with the tested compounds as described above. Then, cell lysates
were prepared using the reagents attached reagents and the assay was performed
according to the detailed description provided by the manufacturer. The extinction was
read at 405 nm using the FLUOstar Omega plate reader (BMG Labtech, Ortenberg,
Germany).
In vitro evaluation of MEK/ERK-mediated phosphorylation activity (MEK/ERK cascade
assay):
To characterize the effect of compound 7k on the activity of MEK/ERK cascade we used
MAP Kinase/Erk Sampler Kit provided by Upstate, Lake Placid, NY and the assay was
performed according to the detailed protocol provided by the manufacturer. In brief, 20
ng of each active phosphorylated MEK (Biaffin, Kassel, Germany) and inactive
nonphosphorylated ERK1, 20 µg of myelin basic protein (MBP) as a substrate and the
tested compound at concentrations ranging from 10-150 µM were mixed in a reaction vial
in a final volume of 50 µL of assay dilution buffer (20 mM MOPS (pH 7.2), 25 mM
sodium-beta-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM
dithithreitol in 50% glycerol). Reaction vials without the tested compound served as
control. As a phosphate donor [γ-32P]ATP cocktail (specific activity 3000 Ci/mmol,
Hartman Analytic, Braunschweig, Germany) was added to the reaction mixture at the
9

amount of 10 µCi/reaction and the assay was performed at 30ºC for 10 min. The
phosphorylated substrate was then separated form residual [γ-32P]ATP using P81
phosphocellulose paper and quantitated by Wallac MicroBeta 1450 scintillation counter
(Perkin-Elmer, WalthamMassachusetts). The assay was performed in quadruplicate and
the results were calculated as percent of control without the tested compound.
In vitro evaluation of pERK1 phosphorylation activity:
To characterize the effect of optimized compound 7p on in vitro activity of activated
pEKR1 we used MAP Kinase/Erk Sampler Kit provided by Upstate, Lake Placid. The
assay was performed according to the manufacturers description as described above with
the following modifications. Reaction mixture contained 20 ng pERK1 and 20 µg BMP
and the optimized compound 7p which was added at the final concentrations ranging
from 0.05-5 µM. Instead of radioactive ATP, non-radioactive ATP was used (Sigma-
Aldrich, St. Louis, MO) was added to the reaction mixture at concentration 0.2 µM.
Following the assay, the remaining ATP was determined with the ATPLite
luciferase/luciferin luminescennt assay system (Perkin-Elmer, Waltham Massachusetts)
and Fluostar Omega plate reader (BMG Labtech) as described in detail in an attached
protocol. The relative activity of pERK was then estimated on a basis of ATP
consumption rate during the assay (decrease of ATP concentration in reaction mixture
following the reaction).
To examine whether inhibitory effect of 7p on pERK1 was related to its competitive
inhibition of substrate binding, we performed a competitive assay with 20 ng pERK1, 5
µM concentration of 7p and different concentrations of MBP ranging from 0.2 to 200
µg/reaction vial.
Evaluation of selectivity of 7p – the studies on various kinases:
To characterize the effect of optimized compound 7p on activity of other kinases, the
ADP-Glo Kinase Assay provided by Promega was employed. The assay was performed
according to the manufacturers description. The solution of an inhibitor was prepared in
buffer A (50 µM). The 5 µL of the solution was preincubated with 5 µL the solution of a
kinase (1 ng) at 25oC at 100 rpm for 10 min. Then, to the reaction mixture was added,
10

containing the substrate (10 – 60 µM), ATP (25µM), buffer A and water. The mixture
was incubated for 1 h at 24oC. Then the ADP-Glo Reagent (25 µL) was added. The
reaction was further incubated for 40 min at 25oC. The Kinase Detection Reagent was
added (25 µL) and the reaction was incubated for additional 30 min. at 25oC and
luminescence was measured.
4.2 Chemistry
Protocol for the enzymatic resolution of rac-4k:
Ester rac-4k (300 mg) was dissolved in 30 ml of solvent (water / Et2O, 8:2, v/v). Porcine
Pancreas Lipase (50.7 mg) was added in one portion and the reaction mixture was stirred
on a shaker at 300 rpm at room temperature for 7 days. Extraction with ethyl acetate,
concentration in vacuo and purification of the resulting residue by flash chromatography
(silicagel, hexane:ethyl acetate) afforded ester (R)-4k and alcohol (S)-5k.
Ester (R)-4k: Yield 52 %. e.e.R(HPLC): 59 %. Rf = 0.21 (hexane/AcOEt; 8:2); mp. 63-
25
64 °C (hexane/AcOEt); [ ]
(c 1.0; CHCl , 59% e.e.); Anal. Calc for C H NO :
3 21 33 4
α
= 2.3
D
C, 69.39%; H, 9.15%; N, 3.85%; found: C, 69.33%; H, 9.27%; N, 3.82%; 1H NMR
(CDCl3, 400 MHz) δ: 0.88-0.92 (t, J = 7.2 Hz, 3H, CH2CH3), 1.20-1.40 (m, 18H,
(CH2)9CH3), 2.11 (s, 3H, CH3CO) 3.78 (s, 3H, CH3O), 5.16-5.22 (m, 2H, ArCH2), 6.20-
6.30 (m, 1H, NH), 6.80-7.30 (m, 4H, ArH). 13C NMR (CDCl3, 100 MHz) δ: 14.1, 21.0,
22.6, 24.7, 29.2, 29.2, 29.4, 29.4, 31.8, 91.9, 42.6, 55.3, 74.2, 114.1, 129.0, 129.9, 159.1,
169.7, 169.7; HPLC: T1 = 7.88, T2 = 11.35 (DAICEL CHIRACEL OD-H, eluent:
hexane/iso-propanol 9:1 (v/v), 1 ml/min).
Alcohol (S)-5k: Yield 35 %. e.e.S (HPLC): 99 %; White crystalls; Rf = 0.60
25
(hexane/AcOEt; 4:6); mp. 112-113 °C (hexane/AcOEt) [ ]
(c 1.0; CHCl , 99%
3
α
= −13.1
D
e.e.) 1H NMR (CDCl3, 400 MHz) δ: 0.85 (t, 3H, J = 6.8 Hz, CH3CH2), 1.20-1.40 (m, 16H,
CH2(CH2)8CH3), 1.59-1.70 (m, 1H, CHCH2(CH2)8), 1.79-1.90 (m, 1H, CHCH2(CH2)8),
11

2.80-2.90 (m, 1H, OH), 3.79 (s, 3H, CH3O), 4.10-4.14 (m, 1H, CH(OH)CH2), 4.36-4.40
(m, 2H, ArCH2NH), 6.75-6.82 (m, 1H, NH), 6.83-7.21 (m, 4H, ArH). 13C NMR (CDCl3,
100 MHz) δ: 14.1, 22.7, 25.0, 29.3, 29.4, 29.5, 31.8, 34.9, 42.6, 55.3, 72.2, 114.0, 129.1,
130.0, 159.0, 173.7. HPLC: TR = 6.27 min., TS = 8.11 min (DAICEL CHIRACEL OD-H,
eluent: hexane/iso-propanol 9:1 (v/v), 1 ml/min.).
Absolute configuration for alcohol 5k was assigned by comparison of the CD spectrum
obtained it with the spectra of similar α-hydroxyamides obtained by EDC-madiated
coupling of α-hydroxyacids with amines. α-Hydroxyacids were prepared from optically
pure, comercially availiable α-aminoacids via diazo compounds. CD spactra are shown
below:
2
1,5
O
1
0,5
OH
0
-0,5
-1
HN
200
210
220
230
240
250
260
270
280
O
-1,5
(nm)
λ
15
10
5
O
OH
0
HN
185
205
225
245
265
-5
O
-10
-15
Mol. CD
λ (nm)
Green line – (R), blue line – (S)
12

2
1.5
1
O
0.5
0
OH
200
210
220
230
240
250
260
270
280
HN
-0.5
-1
O
-1.5
-2
l (nm)
Mol. CD
All the compounds 7 were synthesised using the protocole published by us elswhere.4
The analytical data for all the new compounds is listed below:
7a; White crystals; Rf = 0.42 (hexane/AcOEt; 4:6); mp. 137-138 °C (AcOEt/hexane); 1H
NMR (400 MHz, CDCl3): δ 0.86 (t, 3H, J = 7.0 Hz, CH3CH2), 1.10-1.90 (m, 22H, (CH2)6,
c-Hex), 3.71-3.76 (m, 1H, CHNH), 3.88 (d, 2H, J = 5.4 Hz, gly CH2), 4.31-4.38 (m, 1H,
CHC7H15), 5.12 (s, 2H, CBz CH2), 5.71 (t, J = 5.6 Hz, 1H, NHCBz), 6.33 (d, J = 8.0 Hz,
1H, NH), 6.97 (d, J = 8.0 Hz, 1H, NH), 7.32-7.34 (m, 5H, ArH); 13C NMR (100 MHz,
CDCl3): δ 14.0, 22.6, 24.7, 25.4, 25.5, 29.1, 29.2, 31.7, 32.7, 32.9, 44.4, 48.4, 53.4, 67.1,
128.0, 128.2, 128.5, 136.1, 156.6, 168.9, 170.4; HRMS (ESI, [M+Na]+) 468.2815
(C25H39N3O4Na: 468.2833).
7b; White crystalls; Rf = 0.33 (hexane/AcOEt; 4:6); mp. 104-106 °C (AcOEt/ hexane);
1H NMR (400 MHz, CDCl3): δ 0.86 (t, 3H, J = 6.8 Hz, CH3CH2), 1.10-1.38 (m, 10H,
(CH2)5), 1.52-1.65 (m, 1H, CH2CHNH), 1.75-1.82 (m, 1H, CH2CHNH), 3.77-3.90 (m,
2H, gly CH2), 4.25-4.52 (m, 3H, PhCH2, CHC7H15), 5.02 (s, 2H, PhCH2O), 5.80 (br s,
1H, NHCBz), 7.06-7.42 (m, 11H, ArH, NH); 13C NMR (100 MHz, CDCl3): δ 14.0, 22.6,
25.5, 29.1, 29.2, 31.7, 32.5, 43.4, 44.4, 53.4, 67.1, 127.4, 127.6, 128.0, 128.2, 128.5,
128.6, 136.1, 137.8, 156.6, 169.3, 171.6; HRMS (ESI, [M+Na]+) 476.2523
(C26H35N3O4Na: 476.2520).
13

7c; White crystalls; Rf = 0.38 (hexane/AcOEt; 4:6); mp. 118-119 °C (AcOEt/hexane); 1H
NMR (400 MHz, CDCl3): δ 0.86 (t, 3H, J = 7.0 Hz, CH3CH2), 1.21-1.30 (m, 10H,
(CH2)5), 1.53-1.72 (m, 2H, CH2CH2CHNH), 2.77 (t, 2H, J = 7.2 Hz, PhCH2), 3.38-3.56
(m, 2H, gly CH2), 3.85 (d, 2H, J = 5.3 Hz, gly CH2), 4.32-4.38 (m, 1H, NHCH), 5.08 (s,
2H, CBz CH2), 5.80 (s, 1H, NH), 6.61 (s, 1H, NH), 7.17-7.33 (m, 11H, ArH, NH); 13C
NMR (100 MHz, DMSO-d6 + CDCl3): δ 14.0, 22.6, 25.5, 29.1, 29.2, 31.7, 32.5, 35.5,
40.6, 44.3, 53.3, 67.0, 126.5, 128.0, 128.2, 128.5, 128.5, 128.7, 136.1, 138.6, 156.6, 169.0,
171.5; HRMS (ESI, [M+Na]+) 490.2663 (C26H33N3O5Na: 490.2676).
7d; White crystalls; Rf = 0.58 (hexane/AcOEt; 4:6); mp. 129-130 °C (AcOEt/ hexane);
1H NMR (400 MHz, CDCl3): δ 0.86 (t, 3H, J = 6.8 Hz, CH3CH2), 1.21-1.71 (m, 12H,
(CH2)6), 3.72 (s, 3H, CH3O), 3.83-3.87 (m, 2H, gly CH2), 4.18-4.48 (m, 3H, CH2NH,
CHNH), 5.01 (s, 2H, PhCH2O), 6.78-7.31 (m, 9H, 2ArH), 7.03 (s, 1H, NH); 13C NMR
(100 MHz, CDCl3): δ 14.0, 22.6, 25.5, 29.1, 29.2, 31.7, 32.6, 42.9, 44.4, 53.3, 55.2, 67.0,
113.9, 128.0, 128.1, 128.5, 129.0, 129.9, 136.1, 156.6, 158.9, 169.2, 171.4; HRMS (ESI,
[M+Na]+) 506.2619 (C27H37N3O5Na: 506.2625).
1
7e; White crystalls; Rf = 0.33 (hexane/AcOEt; 4:6); mp. 90-92 °C (AcOEt/hexane); H
NMR (400 MHz, CDCl3): δ 0.86 (t, 3H, J = 6.8 Hz, CH3CH2), 1.10-1.40 (m, 10H,
(CH2)5), 1.52-1.62 (m, 1H, CH2CHNH), 1.72-1.81 (m, 1H, CH2CHNH), 3.76 (s, 3H,
CH3O), 3.79 (s, 3H, CH3O), 3.80-3.91 (m, 2H, gly CH2), 4.25-4.42 (m, 3H, ArCH2,
CHC7H15), 5.07 (s, 2H, PhCH2O), 5.63 (br s, 1H, NHCBz), 6.38-6.42 (m, 3H, ArH), 6.57
(br s, 1H, NH), 6.91 (br s, 1H, NH), 7.11 (d, J = 8.4 Hz, 1H, ArH), 7.30-7.40 (m, 5H,
ArH); 13C NMR (100 MHz, CDCl3): δ 14.0, 22.6, 25.3, 29.1, 29.2, 31.7, 32.8, 39.1, 44.3,
53.2, 55.3, 55.3, 67.1, 98.5, 103.8, 118.2, 128.1, 128.2, 128.5, 130.3, 136.2, 156.5, 158.5,
160.6, 168.7, 171.0; HRMS (ESI, [M+Na]+) 536.2753 (C28H39N3O6Na: 536.2731).
7g; White crystalls; Rf = 0.29 (hexane/AcOEt; 4:6); mp. 163-163 °C (AcOEt/hexane); 1H
NMR (400 MHz, DMSO-d6 + CDCl3): δ 2.76 (dd, 1H, J = 8.4 Hz, J = 13.6 Hz, CH2CH),
2.92 (dd, 1H, J = 5.6 Hz, J = 14.0 Hz, CH2CH), 3.31 (s, 2H, gly CH2), 3.68 (s, 3H,
14

CH3O), 3.69 (s, 3H, CH3O), 4.15-4.19 (m, 2H, PMBCH2NH), 4.38-4.49 (m, 1H,
CHCH2), 4.97 (s, 2H, PhCH2O), 6.70-7.30 (m, 13H, ArH), 7.92-7.93 (m, 1H, NH), 8.23-
8.26 (m, 1H, NH); 13C NMR (100 MHz, DMSO-d6 + CDCl3): δ 36.9, 41.5, 43.5, 54.1,
54.6, 54.7, 65.5, 113.3, 127.4, 127.5, 128.0, 128.3, 129.2, 130.0, 130.6, 136.6, 156.3,
157.6, 158.0, 168.6, 170.4; HRMS (ESI, [M+Na]+) 528.2129 (C28H31N3O6Na: 528.2105).
7h; Yellow oil; Rf = 0.20 (hexane/AcOEt; 4:6); 1H NMR (400 MHz, CDCl3): δ 1.24-1.35
(m, 6H, (CH2)3), 1.41 (s, 9H, CH(CH3)3), 1.57-1.82 (m, 2H, CH2CH2CH(NH2)), 3.04-
3.06 (m, 2H, CH2NH), 3.70-3.75 (m, 1H, CHNH2), 3.76 (s, 3H, CH3O), 4.15-4.45 (m,
4H, CH2Ph, gly CH2), 4.70 (br s, 1H, NH), 5.02 (s, 2H, PhCH2O), 6.81-7.20 (m, 4H,
ArH), 6.85-6.89 (m, 1H, NH); 13C NMR (100 MHz, CDCl3): δ 24.7, 25.9, 28.4, 29.5,
32.2, 40.0, 42.9, 44.4, 53.1, 55.2, 67.0, 79.1, 113.9, 121.5, 126.8, 128.0, 128.1, 128.5,
129.0, 129.9, 136.1, 156,7, 158.9, 169.4, 171.4; HRMS (ESI, [M+Na]+) 593.2924
(C30H42N4O7Na: 593.2955).
7i; White crystalls; Rf = 0.22 (hexane/AcOEt; 4:6); mp. 198-199 °C (AcOEt/hexane) 1H
NMR (400 MHz, CDCl3): δ 0.91-1.66 (m, 11H, c-Hex), 3.76 (s, 3H, CH3O), 3.87 (s, 2H,
gly CH2), 4.26-4.34 (m, 2H, CH2Ph), 5.06 (s, 2H, PhCH2O), 5.52 (s, 1H, NH), 6.49 (s,
1H, NH), 6.76 (s, 1H, NH), 6.82-7.33 (m, 4H, ArH); HRMS (ESI, [M+Na]+) 490.2305
(C26H33N3O5Na: 490.2312).
7k; White crystals; Rf = 0.40 (hexane/AcOEt; 4:6); mp. 141-144 °C; 1H NMR (CDCl3,
400 MHz) δ: 0.82-0.92 (m, 3H, CH3(CH2)8), 1.16-1.86 (m, 16H, CH3(CH2)8), 3.74 (s, 3H,
CH3O), 3.80-3.88 (m, 2H, gly CH2), 4.20-4.50 (m, 3H, ArCH2NH, COCH(NH)CH2),
4.98-5.10 (m, 2H, OCH2Ar), 5.56-5.66 (m, 1H, NH), 6.66-6.74 (m, 1H, NH), 6.74-6.90
(m, 3H, ArH, NH), 7.10-7.20 (m, 2H, ArH), 7.22-7.40 (m, 5H, ArH); 13C NMR (CDCl3,
100 MHz) δ: 14.1, 22.6, 25.5, 29.3, 29.4, 29.5, 31.8, 32.5, 43.0, 53.3, 55.2, 67.2, 114.0,
128.1, 128.2, 128.5, 129.0, 129.9, 136.0, 159.0, 168.9, 171.1; HRMS (ESI, [M + Na]+)
534.2914 (C29H41N3O5Na: 534.2938). Compounds obtained from the products of
25
25
enzymatic reaction: (R) - [ ]
(c 1.0; CHCl ); (S) - [ ]
(c 1.0; CHCl ).
= −8.0
3
α
= +7.6
α
3
D
D
15

7l; Clear oil; Rf = 0.59 (hexane/AcOEt; 4:6); 1H NMR (CDCl3, 400 MHz) δ: 0.80-0.92
(m, 3H, CH3CH2), 1.10-1.95 (m, 16H, (CH2)8), 2.84-2.92 (m, 3H, CH3N), 3.74-3.80 (m,
3H, CH3O), 4.20-4.40 (m, 4H, ArCH2NH, COCH2NH), 4.90-5.40 (m, 1H,
COCH(N)CH2), 5.08-5.14 (m, 2H, OCH2Ph), 6.30-6.42 (m, 1H, NH), 6.48-6.56 (m, 1H,
NH), 6.80-6.86 (m, 2H, ArH), 7.10-7.16 (m, 2H, ArH), 7.24-7.38 (m, 5H, ArH); 13C
NMR (CDCl3, 100 MHz) δ: 14.1, 22.0, 22.6, 26.0, 22.4, 31.3, 33.8, 42.7, 42.9, 55.2, 56.0,
56.8, 67.0, 114.0, 114.0, 128.0, 128.2, 128.5, 128.9, 128.9, 159.0, 169.7, 170.4, 172.0;
HRMS (ESI, [M + Na]+) 548.3117 (C30H43N3O5Na: 548.3095).
7m; White crystals; Rf = 0.22 (hexane/AcOEt; 4:6); mp. 131-134 °C (AcOEt/hexane);
1H NMR (400 MHz, CDCl3): δ 0.87 (t, 3H, J = 7.0 Hz, CH3CH2), 1.10-1.35 (m, 19H,
(CH2)8, CH3CH), 1.52-1.62 (m, 1H, CH2CHNH), 1.72-1.81 (m, 1H, CH2CHNH), 3.73 (s,
3H, CH3O), 4.20-4.44 (m, 3H, gly CH3CH, ArCH2NH), 4.84-5.05 (m, 2H, PhCH2O),
5.60 (d, 1H, J = 6.6 Hz, NHCBz), 6.79-7.38 (m, 11H, 2ArH, 2NH); 13C NMR (100 MHz,
CDCl3): δ 14.1, 18.6, 22.6, 25.5, 29.3, 29.3, 29.4, 29.5, 29.5, 31.8, 32.2, 32.5, 42.9, 42.9,
50.7, 53.3, 55.2, 67.0, 114.0, 128.0, 128.2, 128.5, 128.9, 129.0, 130.0, 136.0, 156.0, 158.9,
171.2, 171.3, 172.4; HRMS (ESI, [M+Na]+) 548.3081 (C30H43N3O5Na: 548.3095).
7n; White crystalls; Rf = 0.46 (hexane/AcOEt; 4:6); mp. 119-122 °C (AcOEt/hexane);
1H NMR (400 MHz, CDCl3): δ 0.85-0.92 (m, 18H, 2 x CH3CH2, 2 x (CH3)2CH), 1.22-
1.90 (m, 38H, 2 x (CH2)8, 2 x (CH3)2CHCH2), 3.71 (s, 3H, CH3O), 3.73 (s, 3H, CH3O),
4.18-4.47 (m, 8H, 2 x leu CH, 2 x C9H19CH, 2 x CH2NH), 4.83-5.05 (m, 4H, 2 x
PhCH2O), 5.54 (d, 1H, J = 8.0 Hz, NHCBz), 5.60 (d, 1H, J = 6.4 Hz, NHCBz), 6.78-7.32
(m, 22H, 2 x 2ArH, 2 x 2NH); 13C NMR (100 MHz, CDCl3): δ 14.1, 21.9, 22.1, 22.6,
22.7, 22.8, 24.6, 24.7, 25.6, 25.6, 29.2, 29.3, 29.3, 29.4, 29.5, 29.5, 31.8, 32.0, 32.4, 41.4,
41.5, 42.8, 42.9, 53.2, 54.0, 55.1, 67.0, 113.9, 127.9, 128.1, 128.4, 129.0, 130.1, 136.0,
136.1, 156.2, 158.8, 158.9, 171.3, 171.3, 172.4, 172.5; HRMS (ESI, [M+Na]+) 590.3543
(C33H49N3O5Na: 590.3564). This compound was obtained as a ca 1:1 mixture of two
diastereoisomers, as judged by the integration of NMR singlets for methoxy group at 3.71
ppm and 3.73 ppm.
16

7o; White crystalls; Rf = 0.42 (hexane/AcOEt; 4:6); mp. 135-140 °C (AcOEt/hexane);
1H NMR (400 MHz, CDCl3): δ 0.86 (t, 3H, J = 7.0 Hz, CH3CH2), 0.88 (t, 3H, J = 7.0 Hz,
CH3CH2), 1.01-1.82 (m, 32H, 2 x (CH2)8), 2.99 (d, 4H, 2 x PhCH2), 3.69 (s, 3H, CH3O),
3.72 (s, 3H, CH3O), 4.18-4.53 (m, 8H, 2 x phe CH, 2 x C9H19CH, 2 x CH2NH), 4.81-
5.00 (m, 4H, 2 x PhCH2O), 5.51 (d, 1H, J = 6.6 Hz, NHCBz), 5.65 (d, 1H, J = 6.6 Hz,
NHCBz), 6.59-7.31 (m, 32H, 2 x 3ArH, 2 x 2NH); 13C NMR (100 MHz, CDCl3): δ14.1,
22.6, 25.4, 25.5, 29.3, 29.4, 29.5, 29.5, 29.5, 31.8, 31.9, 32.0, 32.3, 38.3, 38.5, 42.9, 53.4,
55.1, 55.2, 56.1, 56.8, 67.0, 67.1, 113.9, 114.0, 127.0, 127.1, 127.9, 128.0, 128.1, 128.2,
128.5, 128.5, 128.6, 128.9, 129.0, 129.2, 129.2, 130.1, 136.0, 136.0, 136.0, 136.1, 156.0,
158.8, 158.9, 170.9, 171.0, 171.0, 171.1; HRMS (ESI, [M+Na]+) 624.3391
(C36H47N3O5Na: 624.3407). This compound was obtained as a ca 1:1 mixture of two
diastereoisomers, as judged by the integration of NMR singlets for methoxy group at 3.69
ppm and 3.72 ppm.
7p; White crystalls; Rf = 0.35 (hexane/AcOEt; 5:5); mp. 96-97 °C (AcOEt/hexane); 1H
NMR (200 MHz, CDCl3): δ 0.87 (t, 3H, J = 6.8 Hz, CH3CH2), 1.21-1.30 (m, 14H,
(CH2)7), 1.40-1.82 (m, 2H, CH2CH2CHNH), 2.78 (t, 2H, J = 5.0 Hz, PhCH2), 3.38-3.64
(m, 2H, CH2CH2NH), 3.65-4.00 (m, 2H, gly CH2), 4.22-4.40 (m, 1H, NHCH), 5.10 (s,
2H, CBz CH2), 5.68 (s, 1H, NH), 6.49 (s, 1H, NH), 7.13-7.44 (m, 11H, ArH, NH); 13C
NMR (50 MHz, CDCl3): δ 14.4, 23.0, 25.8, 29.6, 29.8, 29.9, 32.2, 32.6, 35.7, 41.0, 53.8,
67.6, 126.8, 127.9, 128.3, 128.6, 128.9, 128.9, 129.0, 157.1, 169.5, 172.0; HRMS (ESI,
[M+Na]+) 518.2978 (C29H41N3O4Na: 518.2989).
Acknowledgment
This work was supported by project “Biotransformations for pharmaceutical and
cosmetics industry” No. POIG.01.03.01-00-158/09-01 part-financed by the European
Union within the European Regional Development Fund. We also thank to Aleksandra
Stanczak and Monika Lamparska – Przybysz from Celonpharma for determining the
activity of the available kinases.
References and Notes
17

1. Aurelio, L.; Brownlee, R.T.C.; Hughes, A.B. Chem. Rev. 2004, 104, 5823.
2. Huang, Y.; Hang, Y.-B.; Chen, Z.C.; Xu, P.F. Tetrahedron: Asymmetry 2006, 17,
3152.
3. Marastoni, M.; Baldisserotto, A.; Canella, A.; Gavioli, R.; De Risi, C.; Pollini, G.P.;
Tomatis, R. J. Med. Chem. 2004, 47, 1587.
4. Szymanski, W.; Zwolinska, M.; Ostaszewski, R. Tetrahedron 2007, 63, 7647.
5. Szymanski, W.; Ostaszewski, R. Tetrahedron: Asymmetry 2006, 17, 2667.
6. Campbell, S.L.; Khosravi-Far, R.; Rossman, K.L.; Clark G.J.; Der, C.J. Oncogene
1998, 17, 1395.
7. Chang, F.; Steelman, L.S.; Lee, J.T.; Shelton, J.G.; Navolanic, P.M.; Blalock, W.L.;
Franklin, R.A.; McCubrey, J.A.; Leukemia 2003, 17, 1263.
8. Gupta, S.; Plattner, R.; Der, C.J.; Stanbridge, E.J. Mol. Cell. Biol 2000, 20, 9294
9. Adjei, A. A., Cohen, R.B., Franklin, W, Morris, C., Wilson, D., Molina J.R., Hanson,
L.J., Gore, L., Chow, L., Leong, S., Maloney, L., Gordon, G., Simmons, H., Marlow, A.,
Litwiler, K., Brown, S., Poch, G., Kane, K., Haney, J., Eckhardt, S.G.,.; J. Clin. Oncol
2008, 26, 2139.
10. Lorusso P.M., Adjei, A.A., Varterasian, M., Gadgeel, S., Reid, J., Mitchell, D.Y.,
Hanson, L., DeLuca, P., Bruzek, L., Piens, J., Asbury, P., Van Becelaere, K., Herrera, R.,
Sebolt-Leopold, J., Meyer, M.B.,.; J. Clin. Oncol 2005, 23, 5281.
11. Yingchun, L.; Sukumar, S.; Chen, Q.Z.; Keng, Y.F.; Khazak, V.; Illgen, K.; Shabbert,
S.; Weber, L.; Menon, S.R.; Bioorg. Med. Chem. Lett 2004; 14; 3957.
12. Klein, P.J., Schmidt, C.M., Wiesenauer, C.A., Choi, J.N., Gage, E.A., Yip-Schneider,
M.T., Wiebke, E.A., Wang, Y., Omer, C., Sebolt-Leopold, J.S.,.Neoplasia 2006, 8, 1.
13. Maruta, H.; Holden, J.; Sizeland, A.; D’Abaco, G.; J. Biol. Chem 1991, 226, 11661.
14. Młynarczuk-Biały, I.; Roeckmann, H.; Kuckelkorn, U.; Schmidt, B.; Umbreen, S.;
Gołab, J.; Ludwig, A.; Montag, C.; Wiebusch, L.; Hagemeier, C.; Schadendorf D.;
Kloetzel, P.M.; Seifert, U.; Cancer Research 2006, 66, 7598.
Tables
18

Table 1 Optimisation of R1 and R2 fragments (Yields)
No. R1
R2
rac-4
4→5
5→6a
6→7
c-hex
CH3(CH2)6
CH3(CH2)6
CH3(CH2)6
CH3(CH2)6
65% 95% 75% 97%
87% 99% 81% 96%
81% 96% 72% 95%
83% 94% 79% 84%
86% 94% 81% 90%
89% 99% 96% 73%
a
b
c
d
e
f
PhCH2
PhCH2CH2
4-(MeO)-PhCH2
2,4-di-(MeO)-PhCH2 CH3(CH2)6
4-(MeO)-PhCH2
4-(MeO)-PhCH2
4-(MeO)-PhCH2
4-(MeO)-PhCH2
4-(MeO)-PhCH2
4-(MeO)-PhCH2
PhCH2CH2
PhCH2
4-(MeO)-PhCH2 99% 92% 86% 75%
(CH2)5NHBoc 96% 99% 84% 93%
g
h
i
c-Hex
99% 93% 15% 65%
(CH3)2CHCH2 73% 97% 84% 80%
j
(CH2)8CH3
(CH2)8CH3
80% 99% 93% 83%
96% 98% 81% 99%
k
p
a Summary yield over 3 steps;
Table 2 Biological activity of compounds 7a-e*
Cell line
RAS-3T3
NIH-3T3
7a 7b 7c 7d 7e
30 33 20 40 40
40 38 33 40 50
* Results are shown as IC50 (µM)
Table 3 Biological activity of compounds 7f-k*
Cell line
RAS-3T3
NIH-3T3
7d 7f
7g
7h
7i 7j
7k
40 >100 >100 >100 40 >100 19
40 >100 >100 >100 50 >100 55
* Results are shown as IC50 (µM)
Table 4 Biological activity of compounds 7m-o*
Cell line
7k 7m 7n
7o
19 55 50
68
RAS-3T3
19

55 150 >200 >200
* Results are shown as IC50 (µM)
NIH-3T3
20

Figures
100
75
50
25
0
7k
(R)-7k
(S)-7k
10
50
150
Compound concentrations (µM)
Fig 1. In vitro effect of compund 7k racemate and its enantiomers on activity of
MEK/ERK1 kinase cascade. Results are shown as percent MEK/ERK1 activity in the
absence of the tested agents. Each bar shows mean SD from 3 independent experiments.
21

B
100
80
A
60
40
20
0
U0126 U0126 7p
10 µM 40 µM 40 µM
Control
Fig 2.(A) Effect of compound 7p and U0126 on ERK1/2 phosphorylation in RAS-3T3
cells as revealed by Western blot analysis (A) and evaluation by specific ELISA (B).
Western blot analysis and ELISA were performed as described in detail in Material and
Methods. Untreated RAS-3T3 cells served as controls. Figure show results of a
representative experiment.
22

A
B
100
100
80
60
40
20
0
80
60
40
20
0
0.2
20
200
0
0.05
0.5
5
Concentration of MBP (µg)
Concentration of compound 7p (µM)
Fig. 3. In vitro effect of compound 7p on activity of ERK1. (A) Effect of different doses
of compound 7p on ERK1-mediated phosphorylation of BMP. Reactions were performed
at the presence of 20 µg of BMP. Samples without 7p served as control. (B) Evaluation
of ERK1 activity at 5 µM dose of 7p and different concentrations of BMP. As control
served samples without 7p and different corresponding amounts of BMP. Each bar shows
mean SD from 3 independent experiments.
23

Fig 4: The effect of 7p and U0126 on activity of various kinases. The % of activity of a
kinase was measured versus the activity without an inhibitor. Evaluation of the kinases
activities was performed at 10 µM dose of an inhibitor. Bars show mean SD from 2
independent experiments
24

Schemes
Scheme 1. Synthetic methodology.
Scheme 2. Optimisation of R1 and R2 fragments; a) CH2Cl2, rt; b) NaOH, H2O/MeOH; c)
MsCl, Et3N, DMAP, CH2Cl2, 20 °C, 30 min; d) NaN3, DABCO, DMAP, benzo-15-
crown-5, CH2Cl2, 40 °C, 24 h; e) H2, Pd/C, methanol, 4h. f) CBz-glycine, EDC, HOBt,
CH2Cl2, 20 °C, 20 h.
Scheme 3. Optimisation of R3; a) MeNH2, H2O/DMF, 50 °C; b) CBz-glycine, EDC,
HOBt, CH2Cl2, 20 °C, 20 h.
25

Scheme 4. Optimised structure 7p.
Scheme 5. Synthesis of enantiomerically enriched compounds 5k; a) PPL, Phosph. Buff.
pH 7.0 / Et2O, 8:2 (v/v); b) NaOH, MeOH, H2O.
26