Synthesis, biological evaluation, X-ray molecular structure and molecular docking studies of RGD mimetics containing 6-amino-2,3-dihydroisoindolin-1-one fragment as ligands of integrin αiIbβ3

Article abstract of DOI:10.1016/j.bmc.2013.05.019

A series of novel RGD mimetics containing phthalimidine fragment was designed and synthesized. Their antiaggregative activity determined by Born's method was shown to be due to inhibition of fibrinogen binding to α_IIbβ₃. Molecular docking of RGD mimetics to α_IIbβ₃ receptor showed the key interactions in this complex, and also some correlations have been observed between values of biological activity and docking scores. The single crystal X-ray data were obtained for five mimetics.

Full text of DOI:10.1016/j.bmc.2013.05.019

Bioorganic & Medicinal Chemistry 21 (2013) 4646–4661
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, X-ray molecular structure
and molecular docking studies of RGD mimetics containing
6-amino-2,3-dihydroisoindolin-1-one fragment as ligands
q
of integrin
aIIbb3
a
Andrei A. Krysko a, , Georgiy V. Samoylenko , Pavel G. Polishchuk a,b, Marina S. Fonari c,
Victor Ch. Kravtsov c, Sergei A. Andronati a, Tatyana A. Kabanova a, Janusz Lipkowski d,
Tetiana M. Khristova a,b, Victor E. Kuz’min a, Vladimir M. Kabanov a, Olga L. Krysko a, Alexandre A. Varnek b
⇑
a A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Lustdorfskaya Doroga 86, Odessa 65080, Ukraine
b Laboratory of Chemoinformatics, University of Strasbourg, 1 Rue B. Pascal, Strasbourg 67000, France
c Institute of Applied Physics, Academy of Sciences of Moldova, Academiei 5, Chisinau 2028, Republic of Moldova
d Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44, 01 224 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 January 2013
Revised 1 May 2013
Accepted 13 May 2013
Available online 22 May 2013
A series of novel RGD mimetics containing phthalimidine fragment was designed and synthesized. Their
antiaggregative activity determined by Born’s method was shown to be due to inhibition of fibrinogen
binding to aIIbb3. Molecular docking of RGD mimetics to aIIbb3 receptor showed the key interactions in
this complex, and also some correlations have been observed between values of biological activity and
docking scores. The single crystal X-ray data were obtained for five mimetics.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Keywords:
Fibrinogen receptor antagonists
aIIbb3
RGD mimetics
Phthalimidine
Platelet aggregation
1. Introduction
aggregation regardless of the activation pathway.3,4 Inhibitors of
aIIbb3 have proven useful in reducing the risk of periprocedural
Thrombosis is the most important pathological process under-
lying many cardiovascular diseases, which are responsible for ele-
vated mortality worldwide.1 The use of ADP and thromboxane
inhibition does not preclude the formation of thrombus, because
platelets can still be activated by other mechanisms. It gave a stim-
ulus to the development of an entirely separate class of antiplatelet
MI and urgent target vessel revascularization during catheteriza-
tion and have claimed a place in therapy for these indications.5
There exist three different aIIbb3 inhibitors approved for use: abcix-
imab (Eli Lilly & Company, Indianapolis, Indiana), eptifibatide
(Schering-Plough, Kenilworth, New Jersey), and tirofiban (Medi-
cure, Winnipeg, Canada).6 However, the current agents have sev-
eral limitations, including the need for intravenous infusion and,
most important, the induction of thrombocytopenia in some pa-
tients.7 These limitations of above mentioned drugs give evidence
drugs - fibrinogen receptor (integrin aIIbb3) antagonists. During the
platelet activation process, the surface of the platelet transforms its
shape to expose the fibrinogen receptors. These receptors bind to
fibrinogen and Von Willebrand factor, resulting in clot formation
of need in new
aIIbb3 inhibitors.
and clot adherence, respectively.2 Binding of fibrinogen to
aIIbb3
Although, more frequently the design of aIIbb3 antagonists is
on platelets is responsible for securing aggregated platelets to
one another. Thus, blocking these receptors prevents platelet
based on the mimicking of Arg–Gly–Asp (RGD) sequence, alterna-
tive approach represents the use of dodecapeptide sequence.8,9 The
main binding sites of RGD sequence are d-guanidine of arginine
and b-carboxylic group of aspartyl, and for the dodecapeptide se-
quence - lysine amino group and aspartyl carboxylic group, corre-
spondingly. Similarity of these two approaches is evident.
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
In our recent publication, the use of phthalimidine scaffolds for
⇑
Corresponding author. Tel.: +380 308663041; fax: +380 482652012.
E-mail addresses: peptides@paco.net, peptides@physchem.od.ua (A.A. Krysko).
designing potent integrin
aIIbb3 antagonists has been demon-
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.019

A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
4647
strated.10 Herein, we describe the optimization of the novel Arg
surrogates for obtaining RGD mimetics active inhibitors.
This article describes the synthesis of new RGD mimetics con-
taining phthalimidine (2,3-dihydroisoindol-1-one) fragment and
study of their antiaggregative properties. We also discuss the pos-
sibility to use the residues of 4-piperidineacetic, 4-piperidine-4-yl-
butyric, 4-piperidine-4-yl-benzoic, 4-piperazine-1-yl-benzoic, 1,2,
3,4-tetrahydroisoquinoline-7-carboxylic, and 3-piperazine-1-yl-
benzoic acids as Arg surrogates for RGD mimetics design.
Figure 1. View of 5b. ORTEP diagram with the numbering scheme. The thermal
ellipsoids are of 50% probability level, with hydrogens as spheres of arbitrary
diameter.
2. Results and discussion
2.1. Synthesis
a
H
2N
HN
N
O
O
Description of the synthesis of 6-amino-2,3-dihydroisoindolin-
1-one building blocks 5a–d was given in a previous report
(Scheme 1). The key step was the formation of the 6-amino-2,3-
dihydroisoindolin-1-one building blocks 5a–d by reduction of
phthalimides 1a–d using zinc amalgam. As a result of nitration of
the compounds 2a–d, there were obtained nitro-derivatives 3a–d
followed by their reduction by H2/Pd(C). Regioselectivity of nitra-
tion reaction is expected. The X-ray analysis data obtained for
the compound 5b is a confirmation.
The view of molecular structure of 5b is shown in Figure 1. The
molecule has a rather flat skeleton as evidenced by the dihedral an-
gle of 8.8° between the planes of almost coplanar nonhydrogen
atoms of methylpropionate chain and phthalimide core. The geo-
metric parameters of planar aminophthalimide core are in line
with the related 3-(1-oxo-1,3-dihydroisoindol-2-yl)propionic
acid,11 but these molecules differ by the molecular shape as far
as the latter one has an angular conformation.
Homologation of Boc protected piperidine-4-carboxylic acid
gave the 1-Boc-4-piperidineacetic acid (6), by a similar method re-
ported in the literature.12 Initial Boc derivatives of the 4-piperi-
dine-4-yl-butyric (7)13 and 4-piperidine-4-yl-benzoic (8)14 acids
have been synthesized using previously published methods. Iso-
meric methyl esters of the 4-piperazine-4-yl-benzoic (9a)15 and
3-piperazine-4-yl-benzoic (9b) acids were made using methyl es-
ters of aminobenzoic acids and bis-(2-chloroethyl)amine. Boc-pro-
tection of the compounds 9a and 9b with Boc2O resulted in the
methyl esters 10a and 10b, respectively. Subsequent saponification
of ester groups of compounds 10a and 10b yielded the target acids
11a,b (Scheme 2).
Precursor compound 14 was prepared in three steps in contrast
to our previously published route for four-steps synthesis.16 Acyl-
ation of 2-acetyl-1,2,3,4-tetrahydroisoquinoline (12) with oxalyl
chloride at the presence of aluminum chloride gave 1-acetyl-
1,2,3,4-tetrahydroisoquinoline-7-carboxylic acid (13). Further
O
10a,b
12
O
9a,b
N
b
O
O
O
O
c, d
N
N
N
OH
O
11a,b
O
O
e
N
N
OH
13
O
O
O
f,b,d
O
N
OH
14
O
O
Scheme 2. Synthesis of the acids 11 and 14. Reagents: (a) HCl⁄NH(CH2CH2Cl)2,
K2CO3, BuOH, reflux, ꢀ177 h; (b) Boc2O, room temperature, overnight; (c) NaOH,
H2O 40 °C, 6 h; (d) HCl, H2O; (e) (COCl)2, AlCl3; (f) NaOH, H2O.
removal of the acetyl group and reaction with Boc2O resulted in
the acid 14 (Scheme 2). The structure of compound 14 has been
confirmed by data of X-ray analysis (Fig. 2).
The molecular structure 15 is shown in Figure 2. The molecule
has an angular shape with the aza-cycle of the 1,2,3,4-tetrahydro-
isoquinoline core being in a half-chair conformation with the N1
atom deviating at À0.574(3) Å from the mean plane of cyclic C-
atoms, the rms deviation of fitted carbon atoms being 0.056 Å.
Condensation of acids 6–8, 11a,b and 14 with amines 5a–d has
been conducted using the HBTU or HATU (Scheme 3). Subsequent
saponification of ester groups of the compounds 15a–u and
O
R
R
R
( )n
b
a
N
O
N
O
N
O
( )n
( )n
O2N
OH
O
OH
O
OH
1a-d
O
3a-d
2a-d
c
R
R
N
O
O
d
N
O
O
( )n
O2N
( )n
H2N
O
O
4a-d
5a-d
R = H, CH3; n = 0, 1, 2
Scheme 1. Synthesis of aminophthalimidines. Reagents: (a) Zn (Hg), HCl, reflux, 4 h; (b) HNO3, H2SO4, À5 °C, 6 h; (c) MeOH (H2SO4), reflux, 3 h; (d) H2/Pd (C), MeOH, room
temperature, 7 h.

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A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
phthalimidine core and anti-conformation with regard to the
benzoyl moieties as the torsion angles O2–C9–N2–C6 and C10–
C9–N2–C6 indicate falling in the ranges 0.6(8)–8.7(9)° and
171.1(6)–179(1)°, respectively (Table 1). The molecules differ by
mutual orientations of the above-mentioned planar molecular
moieties, by the orientation of piperidine or piperazine fragment
with respect to the benzoyl moieties, and carboxylic tails with
respect to the indolyl core. Moreover, the different mutual arrange-
ment (cis- or trans-) of the amide and indolyl carbonyl oxygen
atoms O1 and O2 should be mentioned. The conformational flexi-
bility and essential variation of cross-molecule distance between
the distal cyclic N and carboxylic O-atoms in the wide range
16.297–21.434 Å, Table 1, indicate the possibility to fine tuning
substrate-receptor interactions.
Figure 2. View of 15. ORTEP diagram with the numbering scheme. The thermal
ellipsoids are of 50% probability level, with hydrogens as spheres of arbitrary
diameter.
2.3. In vitro biology
elimination of Boc-protective groups yielded the target mimetics
17a–u. Mimetics 17l–q were obtained only as racemic mixtures
in order to reveal potent compounds and to determinate general
characteristics of structure–activity relationships.
Functional activity was determined by measuring the inhibition
of ADP induced platelet aggregation in human platelet-rich plasma
(PRP) by Born’s method.17 Mode of action for some compounds was
subsequently revealed in vitro by measuring the ability of com-
pounds to inhibit the binding of fluoresceinisothiocyanate-labeled
2.2. X-ray structure
fibrinogen (FITC-Fg)18 to
a
IIbb3 (in a suspension of human washed
platelets).19 Experimental data (Table 2) evidently show high affin-
ities of the compounds 17 for IIbb3. RGDS peptide and Tirofiban
were used as standard inhibitors.
Fortunately, single crystals of target compounds were grown
from water, and X-ray data were obtained for five mimetics,
17c,h,i,j,t. They were fixed in the solid crystalline state as either tri-
fluoroacetate or chloride salt with the compositions 17c,h and
17i,j,tÁH2O. After 2 h of boiling of aqueous solutions of compounds
17c and 17h and then their cooling to room temperature, single
crystals were grown. They were identified as the hydrates of zwit-
terionic forms, 17c-TfaOHÁ5H2O and 17h-TfaOHÁ2H2O. The ORTEP
drawings for the formula units are shown in Figure 3. All the
mimetics molecules bear three planar fragments, the benzoyl ring,
the 1-oxo-1,3-dihydroisoindolyl core with the equivalent bond
lengths being similar within themselves, in the parent 5b and in
the closely related 3-(1-oxo-1,3-dihydroizoindol-2-yl)propionic
acid,11 and the amide bridge between these moieties. The amide
bridge is always fixed in the syn-conformation with regard to the
a
We first investigated the SAR of compounds 17a–e, containing a
fragment of (1-oxo-1,3-dihydroisoindol-2-yl)acetic acid. These
compounds showed low in vitro antiaggregative activity, except
for the leader of the group, mimetic 17d containing 4-piperazine-
4-yl-benzoyl, as Arg isostere. Homologation of C-terminal template
resulted in increased activity for five compounds 17f–j containing
the same surrogates of Arg. Here again was the leading mimetic
17i, containing a residue of 4-piperazine-4-yl-benzoic acid. Lead-
ing indicators of antiaggregative activity of compounds containing
4-piperazine-4-yl-benzoyl, forced us to test 3-piperazine-4-yl-ben-
zoyl derivative 17k. It was found that the compound 17k was five
times less active than its isomer 17i. Further increase of C-terminal
R
a
N
O
O
oc
B
OH
6-8, 11a,b, 14
Aa
O
Aa
( )n
N
H
O
O
15a-u
b
R
N
O
O
Aa
( )n
N
H
c
OH
O
O
R
N
O
16a-u
O
HX*H
Aa
( )n
N
H
OH
O
17a-u
O
N
N
O
,
N
Aa
,
=
,
O
N
N
N
O
N
N
,
,
O
n = 0 - 2; HX = HCl, CF3COOH
Scheme 3. Synthesis of RGD mimetics. Reagents: (a) NEt3, HBTU, 5a–d, room temperature, overnight; (b) 1 M NaOH, H2O, MeOH, room temperature, overnight, 1 M HCl; (c)
CH2Cl2, HCl gas, 0 °C, 1 h or CH2Cl2, TFA, 0 °C, 2 h.

A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
4649
Figure 3. ORTEP diagram with the numbering scheme for 17c-TfaOHÁ5H2O (a), 17c (b), 17h-TfaOHÁ2H2O (c), 17h (d), 17iÁH2O (e), 17jÁH2O (f), and 18tÁH2O (g). The thermal
ellipsoids are of 30% probability level, with hydrogens as spheres of arbitrary diameter. Only major positions are shown for the disordered fragments.
residue length by another one carbon atom led to a decrease in
aggregative properties. Surprise for us was the value of IC50 for
compound 17r. Mimetic 17r was more active than the mimetic
17b, these two compounds have the same number of atoms
between the N- and C-terminals. Finally, replacing C-terminal frag-
ment b-alanine with b-methyl-b-alanine led to compounds 17l–p.

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A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
Table 1
Main geometrical parameters for 17 entities in studied compounds
Compounds
Geometric parameter
a
b
c
d
e
/
(°)
/
(°)
/
(°)
/
(°)
/ (°)
3
f/4(°)
gr (Å)
1
2
1
2
17c-TfaOHÁ5H2O
17c
À2.5(3)
4.4(6)
176.8(2)
84.87(5)
87.5(1)
70.53(9)
61.1(7)
56.06(8)
3.8(3)
26.51(8)
23.7(2)
4.9(2)
15.3(9)
22.3(1)
29.9(2)
16.6(3)
7.48(6)
79.1(2)
59.7(3)
10.3(3)
9.8(9)
8.6(6)
75.8(2)
15.5(1)
16.466(2) 18.377(2)
17.256(5) 18.718(5)
18.913(3) 19.6857(3)
18.05(3) 19.10(3)
16.297(4) 16.885(4)
16.803(7) 17.091(7)
20.235(7) 21.434(7)
À174.3(3)
39.7(1)
45.1(1)
65.3(4)
17.3(1)
30.9(2)
15.8(3)
17h-TfaOHÁ2H2O
À1.7(4)
175.6(3)
17h
3(3)
179(1)
17iÁH2O
2.2(4)
À175.3(2)
À179.5(4)
171.1(6)
17jÁH2O
À0.6(8)
17tÁH2O
À8.7(9)
80.2(2)
a
b
c
d
e
f
Torsion angle O2–C9–N2–C6.
Torsion angle C10–C9–N2–C6.
Dihedral angle between the average planes through the terminal piperydine/piperazine ring and phenyl ring, C10/C11/C12/C13/C14/C15.
Dihedral angle between the average planes through the phenyl ring, C10/C11/C12/C13/C14/C15 and amide bridge, C10/C9/O2/N2/C6.
Dihedral angle between the average planes through the amide bridge, C10/C9/O2/N2/C6 and phthalimidine core, C1 > C8/N1/O1.
Dihedral angle between the average planes through the phthalimidine core, C1 > C6/N1/O1 and carboxylic group, defined by C,O,O atoms.
Cross-molecule distance between the cyclic N and carboxylic O-atoms.
g
Effect of CH3 group is ambiguous. In two cases, antiaggregative
activity observed for mimetics containing b-methyl-b-alanine frag-
ment (17o and 17q) was lower than for unsubstituted ones (17i
and 17k). As for methylated/unmethylated pairs 17m/18g and
17n/17h, their antiaggregative activities were nearly equal. Posi-
tive effect of methyl group was observed only for compounds 17l
and 17p compared to their unsubstituted analogs 17f and 17j, cor-
respondingly. Compound 17p possessed the highest antiaggrega-
tive activity among all compounds synthesized in this study.
topological length between Asp- and Arg-mimetic parts, can be ex-
plained by different protein–ligand interaction patterns. The car-
boxylic acid group of 18r is buried deeper in the receptor pocket
and interacts with bAsn215 as well as with MIDAS, and carbonyl
group of 2,3-dihydroisoindol-1-one moiety forms H-bond with
OH-group of
aTyr190. On the other hand, carboxylic acid group
of 17b interacts only with MIDAS, whereas carbonyl group of 4-
piperidin-4-yl-butanoyl forms H-bond with water molecules coor-
dinated to aAsp232 (Fig. 5, compounds 17b and 17r). Comparison
of the docking pose of 17i with that of 17d shows that 17i is char-
acterized by bent conformation whereas 18d adopts almost linear
form. Furthermore, 17i is characterized by higher docking score va-
lue compared to 17d (38.6 vs 36.4).
2.4. Molecular docking
Molecular docking studies have been performed in order to
give a microscopic insight into experimentally observed struc-
ture–property relationship. FlexX tool from LeadIT package has
been used.20 The X-ray structure of complex of Tirofiban with
3. Conclusion
headpiece of
aIIbb3 integrin (2VDM) has been taken from Protein
This paper is devoted to rational design of the
antagonists. It has been demonstrated that RGD mimetics contain-
ing 2,3-dihydroisoindol-1-one can be effificiently used as IIbb3
antagonists and platelet aggregation inhibitors through appropri-
ate structural modulation. In particular, modification of the Arg
aIIbb3 integrin
Data Bank.21 Structure of the pocket has been prepared and four
water molecules have been kept in the cavity: two water mole-
cules, coordinated with Mg2+ metal ion-dependent adhesive site
a
(MIDAS) of the
nated with Asp232 residue of the
pounds have been docked in the prepared pocket. Because the
interaction of ligands of IIbb3 integrin with Asp224 and MIDAS
are essential for compound binding and activity, only best poses
which follow these requirements have been taken for further
analysis.21
a
IIbb3 integrin, and two water molecules coordi-
a
IIb-subunit. Investigated com-
surrogates represents an attractive way of optimizing
aIIbb3
integrin ligands. It can be summarized that the most preferable
structural features for high antiaggregative activity are tetrahydro-
isoquinoline and b-methyl-b-alanine as Arg and Asp bioisosteres,
respectively. Combination of these fragments within the given
phthalimidine series leads to the best prospective inhibitor of
platelet aggregation. The most active compounds identified display
favorable biological properties which are promising for further
development of antithrombotics.
a
a
A reasonable correlation between affinity for aIIbb3 and docking
score has been observed (RSpearman = 0.72, Fig. 4) for a small subset
of compounds for which the activity values were available. Antiag-
gregative activity poorly correlates with docking score (RSpearman = 0.35).
However, exclusion of one outlier (compound 17u) significantly
improves the correlation (RSpearman = 0.52, Fig. 4).
Discovered correlations suggest that docking score can be used
for elucidation of antiaggregative activity of the compounds. Thus,
higher antiaggregative potency of 17f–j compared to 17a–e could
be explained by the fact that the former form additional H-bonds
to OH-group of Tyr190 or to water molecules connected with
4. Experiment
4.1. Chemistry
Melting points are uncorrected. 1H and 13C NMR spectra were
recorded on Bruker AVANCE-II-400 (at 400 MHz for proton), or
Bruker Avance DRX 500 (at 500 MHz for proton and 125 MHz for
carbon) spectrometers with chemical shifts in ppm with the inter-
nal TMS as a standard. Electron ionization (EI) and fast-atom bom-
bardment (FAB) mass spectra were recorded on a VG Analytical VG
70-70EQ instrument. FAB spectra were performed equipped with
an argon primary atom beam, and an m-nitrobenzyl alcohol matrix
was utilized. High-resolution mass spectra (HRMS) were recorded
under FAB conditions. The purity was measured by HPLC
aAsp232, whereas the latter cannot form hydrogen bonds to OH-
group of Tyr190 due to the steric reasons (Fig. 5, compounds
a
17e and 17j).
Compounds 17s,t resulted from the increase of length of the
Asp-mimetic part of 17g,h, are too big compared to the cavity
and, therefore they do not interact efficiently with the protein res-
idues (Fig. 5, compounds 17h and 17t).The difference between
activity of the compounds 17b and 17r, which have the same

A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
4651
M)
Table 2
Biological properties of RGD mimetics 17, RGDS peptide and Tirofiban
Compounds
17a
HX
Aa
n
0
0
0
0
R
IC50, (PRP)a
(l
M)
IC50, (FITC-Fg/a (l
IIbb3)b
HN
O
TFA
TFA
TFA
TFA
H
H
H
H
51 11.0
66.0 9.0
24.0 3.0
3.3 0.5
––
HN
O
17b
––
O
O
17c
0.27 0.06
––
HN
HN
17d
N
O
17e
HCl
0
H
120.0 20.0
1.2 0.1
HN
HN
HN
O
17f
17g
17h
17i
TFA
TFA
TFA
TFA
1
1
1
1
H
H
H
H
7.4 1.4
5.9 0.6
––
O
0.0055 0.009
0.0068 0.0012
––
O
O
9.6 1.9
HN
HN
0.54 0.06
N
N
O
17j
HCl
TFA
1
1
H
H
1.1 0.1
2.7 0.5
0.0065 0.0005
––
HN
HN
O
O
17k
HN
HN
O
17l
TFA
HCl
HCl
HCl
1
1
1
1
CH3
CH3
CH3
CH3
2.7 0.6
5.4 1.0
––
17m
17n
17o
0.35 0.03
––
O
O
6.2 1.2
HN
HN
3.74 0.51
0.037 0.08
N
N
O
17p
17q
HCl
HCl
1
1
CH3
CH3
0.086 0.007
62.0 5.0
0.0065 0.0012
––
HN
HN
O
O
HN
HN
O
17r
17s
17t
TFA
TFA
TFA
2
2
2
H
H
H
1.8 0.3
51.0 3.0
––
––
––
O
410.0 60.0
HN
O
17u
HCl
2
H
330.0 50.0
––
HN
RGDS
31.0 2.0
13.0 1.6
Tirofiban
0.032 0.004
0.0024 0.0004
a
Concentration required to reduce ADP-induced human platelet aggregation response by 50%. The IC50 values are expressed as the average of at least three determinations.
The average error for the IC50 determinations was 15%.
b
Concentration required to reduce binding of FITC-Fg to aIIbb3 on the suspension of washed human platelets by 50%. The IC50 values are expressed as the average of at least
three determinations. The average error for the IC50 determinations was 15%.
conducted on an Shumadzu system (System Controller CBM-20A,
two pumps LC-8A and Photo-diode Array detector SPD-M20A)
4.1.1. General procedure for a preparation of compound 2
24.5 g of mossy zinc was amalgamated with 1 g of metallic mer-
cury and 1 M HCl solution (100 ml) in water. The suspension was
shaken for 5 min, and then the aqueous layer was discarded. The
zinc was covered with 24.5 ml of concentrated HCl and to this
was added 0.0488 mol of compound 1. The mixture was heated
to boiling for 0.5 h. At this stage, it is necessary to keep the reaction
temperature under control (at rapid foaming, heating was termi-
nated and reaction mixture was shortly cooled externally). After
all the compound 1 was dissolved the mixture was boiled under re-
using a Hypersil GOLD 3
aQ 3
monitored by TLC (silica gel 60 F254, Merck).
l
m (4.6 Â 150 mm) or Hypersil GOLD
l
m (4.6 Â 150 mm) column. The progress of reactions was
The compound 11a was prepared from 4-aminobenzoic acid
methyl ester by the procedure. The method of synthesis of 11b
from 3-aminobenzoic acid methyl ester not significantly differ
from the procedure for compound 11a. The acids 7, 8 and 14 have
been synthesized using previously published method.

4652
A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
Figure 4. Plots of affinity for
aIIbb3 of 17c,e,g,h,j,m,o,p and antiaggregative activity of 17a–u against docking score.
17e
17j
17h
17t
17r
17b
17i
17d
Figure 5. Docking poses and interactions of compounds 17b,d,e,h–j,r,t inside the
a
IIbb3 receptor cavity.
flux for 4 h. The hot solution was decanted from undissolved zinc
and allowed to cool. white crystalline solid precipitated
and was collected. Recrystallization from water to give pure com-
4.1.1.1. (1-Oxo-1,3-dihydroisoindol-2-yl)acetic acid (2a). Mp =
177–179.5 °C; 1H NMR d (500 MHz, d6-DMSO) 4.30 (s, 2H), 4.53
(s, 2H), 7.50–7.53 (m, 1H), 7.56–7.58 (m, 2H), 7.68 (d, J = 7.4 Hz,
1H), 12.93 (s, 1H); MS (EI) m/z: 191.
A
pound 2.

A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
4653
4.1.1.2. 3-(1-Oxo-1,3-dihydroisoindol-2-yl)propionic acid (2b).
Mp = 113–116 °C; 1H NMR
(500 MHz, d6-DMSO) 2.62 (t,
(s, 2H), 7.90 (d, J = 8.3 Hz, 1H), 8.33 (d, J = 2.0 Hz, 1H), 8.46 (dd, J = 8.3,
2.0 Hz, 1H); MS (EI) m/z: 264.
d
J = 7.0 Hz, 2H), 3.74 (t, J = 7.0 Hz, 2H), 4.50 (s, 2H), 7.47–7.50
(m, 1H), 7.60 (d, J = 3.6 Hz, 2H), 7.68 (d, J = 7.3 Hz, 1H), 12.34
(br s, 1H); MS (EI) m/z: 205.
4.1.3.3. 3-(6-Nitro-1-oxo-1,3-dihydroisoindol-2-yl)butyric acid
methyl ester (4C). Mp = 125–126 °C; 1H NMR d (500 MHz, d6-
DMSO) 1.32 (d, J = 6.8 Hz, 3H) 2.76 (ddd, J = 35.4, 15.2, 7.3 Hz, 2H)
3.57 (s, 3H) 4.59–4.67 (m, 3H) 7.91 (d, J = 8.3 Hz, 1H) 8.34 (s, 1H)
8.46 (dd, J = 8.3, 2.2 Hz, 1H); MS (EI) m/z: 278.
4.1.1.3. 3-(1-Oxo-1,3-dihydroisoindol-2-yl)butyric acid (2C).
Mp = 205.5–206 °C; 1H NMR
d (500 MHz, d6-DMSO) 1.28 (d,
J = 6.6 Hz, 3H), 2.64 (ddd, J = 31.4, 15.3, 7.3 Hz, 2H), 4.46 (dd,
J = 28.4, 17.6 Hz, 2H), 4.62 (dt, J = 13.9, 6.6 Hz, 1H), 7.47–7.50 (m,
1H), 7.60–7.61 (m, 2H), 7.67 (d, J = 7.6 Hz, 1H), 12.26 (br s, 1H); MS
(EI) m/z: 219.
4.1.3.4. 4-(6-Nitro-1-oxo-1,3-dihydroisoindol-2-yl)butyric acid
methyl ester (4d). Mp = 86–87 °C; 1H NMR d (500 MHz, d6-
DMSO) 1.90 (dt, J = 13.8, 6.7 Hz, 2H), 2.37 (t, J = 7.0 Hz, 2H), 3.55 (s,
3H), 3.59 (t, J = 6.7 Hz, 2H), 4.65 (s, 2H), 7.90 (d, J = 8.3 Hz, 1H), 8.34
(s, 1H), 8.46 (d, J = 8.3 Hz, 1H); MS (EI) m/z: 278.
4.1.1.4. 4-(1-Oxo-1,3-dihydroisoindol-2-yl)butyric acid (2d).
Mp = 125–127 °C; 1H NMR
d (400 MHz, d6-DMSO) 1.8 (dt,
J = 14.2, 7.2 Hz, 2H) 2.2 (t, J = 7.3 Hz, 2H) 3.5 (t, J = 7.0 Hz, 2H) 7.5
(m, J = 7.9, 7.9, 4.0 Hz, 1H) 7.6 (m, J = 3.9 Hz, 2H) 7.7 (d, J = 7.6 Hz,
1H) 12.1 (br s, 1H); MS (EI) m/z: 219.
4.1.4. General procedure for a preparation of compound 5
The nitro compound 4 (0.015 mol) dissolved in methanol
(100 ml) was subjected to catalytic hydrogenation at room temper-
ature for 7 h in the presence of 3% palladium on carbon (1 g).
The filtered solution was then evaporated in vacuo to give com-
pound 5.
4.1.2. General procedure for a preparation of compound 3
Compound 2 (0.0252 mol) was dissolved in concentrated H2SO4
(37.7 ml) at 0 °C, and to this solution was added HNO3 (12.7 ml,
d = 1.5) at À10 °C. The mixture was stirred for 6 h at À5 °C. The
reaction mixture was poured onto crushed ice (200 g). A crystalline
solid precipitated and was collected. The filtered product was
recrystallized from methanol.
4.1.4.1. (6-Amino-1-oxo-1,3-dihydroisoindol-2-yl)acetic acid
methyl ester (5a). Mp = 142–143 °C; 1H NMR d (400 MHz, d6-
DMSO) 3.68 (s, 3H), 4.32 (s, 2H), 4.34 (s, 2H), 5.38 (s, 2H), 6.83 (dd,
J = 2.1 Hz, 8.1 Hz, 1H), 6.87 (d, J = 2.1 Hz, 1H), 7.23 (d, J = 8.1 Hz,
1H); MS (EI) m/z: 220.
4.1.2.2. (6-Nitro-1-oxo-1,3-dihydroisoindol-2-yl)acetic acid (3a)
Mp = 222–223 °C; 1H NMR d (400 MHz, DMSO-d6) 4.33 (s, 2H), 4.68
(s, 2H), 7.91 (d, J = 8.3 Hz, 1H), 8.37 (d, J = 2.1 Hz, 1H), 8.48 (dd,
J = 2.1 Hz, 8.3 Hz, 1H); MS (EI) m/z: 219.
4.1.4.2. 3-(6-Amino-1-oxo-1,3-dihydroisoindol-2-yl)propionic
acid methyl ester (5b). Mp = 147–148.5 °C; 1H NMR
d
(400 MHz, d6-DMSO) 2.66 (t, J = 7.1 Hz, 2H), 3.61 (s, 3H), 3.71 (t,
J = 7.1 Hz, 2H), 4.28 (s, 2H), 5.30 (s, 2H), 6.79 (dd, J = 2.1 Hz,
J = 8.0 Hz, 1H), 6.82 (d, J = 2.1 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H); MS
(EI) m/z: 234.
4.1.2.3.
3-(6-Nitro-1-oxo-1,3-dihydroisoindol-2-yl)propionic
acid (3b). Mp = 196.5–197 °C; 1H NMR d (400 MHz, DMSO-d6)
2.65 (t, J = 7.0 Hz, 2H), 3.77 (t, J = 7.0 Hz, 2H), 4.67 (s, 2H), 7.90 (d,
J = 8.3 Hz, 1H), 8.33 (d, J = 2.1 Hz, 1H), 8.45 (dd, J = 2.1 Hz,
J = 8.3 Hz, 1H); MS (EI) m/z: 250.
4.1.4.3. 3-(6-Amino-1-oxo-1,3-dihydroisoindol-2-yl)butyric acid
methyl ester (5C). Mp = 86–86.5 °C; 1H NMR
d (500 MHz,
d6-DMSO) 1.25 (d, J = 6.8 Hz, 3H), 2.68 (ddd, J = 27.0, 15.1, 7.3 Hz,
2H), 3.56 (s, 3H), 4.23 (dd, J = 24.5, 16.4 Hz, 2H), 4.58 (dt, J = 13.9,
6.8 Hz, 1H), 5.30 (s, 2H), 6.79 (dd, J = 8.1, 1.5 Hz, 1H), 6.82 (s, 1H),
7.20 (d, J = 8.1 Hz, 1H); MS (EI) m/z: 248.
4.1.2.4. 3-(6-Nitro-1-oxo-1,3-dihydroisoindol-2-yl)butyric acid
(3C). Mp = 194–195 °C; 1H NMR d (500 MHz, d6-DMSO) 1.31 (d,
J = 6.8 Hz, 3H), 2.67 (ddd, J = 36.9, 15.5, 7.3 Hz, 2H), 4.58–4.68 (m,
3H), 7.90 (d, J = 8.3 Hz, 2H), 8.33 (d, J = 1.7 Hz, 1H), 8.46 (dd, J = 8.3,
1.7 Hz, 1H), 12.31 (s, 1H); MS (EI) m/z: 264.
4.1.4.4. 4-(6-Amino-1-oxo-1,3-dihydroisoindol-2-yl)butyric acid
methyl ester (5d). Mp = 97–98 °C; 1H NMR
d (400 MHz,
4.1.2.5. 4-(6-Nitro-1-oxo-1,3-dihydroisoindol-2-yl)butyric acid
(3d). Mp = 153–155 °C; 1H NMR d (500 MHz, d6-DMSO) d ppm
1.87 (dt, J = 13.8, 7.0 Hz, 2H) 2.28 (t, J = 7.0 Hz, 2H) 3.59 (t,
J = 7.0 Hz, 2H) 4.65 (s, 2H) 7.90 (d, J = 8.3 Hz, 1H) 8.34 (s, 1H)
8.46 (d, J = 8.0 Hz, 1H) 12.07 (s, 1H); MS (EI) m/z: 264.
d6-DMSO) 1.82 (dt, J = 14.2, 7.2 Hz, 2H), 2.30 (t, J = 7.2 Hz, 2H),
3.46 (t, J = 7.2 Hz, 2H), 3.52 (s, 3H), 4.23 (s, 2H), 5.28 (s, 2H), 6.76
(dd, J = 8.1, 2.1 Hz, 1H), 6.81 (d, J = 2.1 Hz, 1H), 7.17 (d, J = 8.1 Hz,
1H); MS (EI) m/z: 248.
4.1.5. 1-Boc-4-piperidineacetic acid (6)
4.1.3. General procedure for a preparation of compound 4
Compound 3 (0.0169 mol) was dissolved in methanol (50 ml),
and to this solution was added concentrated H2SO4 (0.5 ml). The
reaction solution was refluxed for 3 h. The solvent was removed
via evaporation in vacuo, and the residue was triturated with
water. A solid precipitated and was collected. The filtered product
was recrystallized from methanol.
1-Boc-piperidine-4-carboxylic acid (2.29 g, 10 mmol) was dis-
solved in anhydrous THF (25 ml) under argon. The solution was
cooled to À15 °C, and triethylamine (1.4 ml, 10 mmol), and then
isobutyl chloroformate (1.3 ml, 10 mmol), were added. The reactor
was protected from light. After 30 min, an ethereal solution of
diazomethane was added. The mixture was allowed to warm to
room temperature for 2 h without stirring and was left overnight.
The reaction solution was diluted with chloroform (final volume
À200 ml). The excess of diazomethane was destroyed by addition
of few drops of acetic acid, and saturated aqueous solution of
NaHCO3 (10 ml) was added carefully. The aqueous layer was sepa-
rated, and the organic layer was washed with saturated aqueous
NaCl (10 ml). The organic layer was dried over anhydrous Na2SO4,
filtered, and the solvent was evaporated in vacuo to dryness. The
crude product, the diazoketone, was used directly in the next step.
4.1.3.1. (6-Nitro-1-oxo-1,3-dihydroisoindol-2-yl)acetic acid methyl
ester (4a). Mp = 113–115 °C; 1H NMR d (400 MHz, d6-DMSO) 3.70
(s, 3H), 4.46 (s, 2H), 4.70 (s, 2H), 7.94 (d, J = 8.3 Hz, 1H), 8.39 (d,
J = 2.1 Hz, 1H), 8.50 (dd, J = 2.1 Hz, 8.3 Hz, 1H); MS (EI) m/z: 250.
4.1.3.2. 3-(6-Nitro-1-oxo-1,3-dihydroisoindol-2-yl)propionic acid
methyl ester (4b). Mp = 150–152 °C; 1H NMR d (400 MHz, d6-
DMSO) 2.74 (t, J = 7.0 Hz, 2H), 3.62 (s, 3H), 3.80 (t, J = 7.0 Hz, 2H), 4.67

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A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
The diazoketone was dissolved in THF (40 ml), and water
4.0 Hz, 2H), 1.24 (dd, J = 14.9, 6.6 Hz, 2H), 1.39 (s, 9H), 1.41–1.50
(m, 1H), 1.59–1.67 (m, 4H), 2.33 (t, J = 7.3 Hz, 2H), 2.60–2.75 (m,
2H), 3.69 (s, 3H), 3.92 (d, J = 11.0 Hz, 2H), 4.39 (s, 2H), 4.46 (s, 2H),
7.54 (d, J = 8.2 Hz, 1H), 7.72 (dd, J = 8.2, 1.8 Hz, 1H), 8.08 (d,
J = 1.8 Hz, 1H), 10.13 (s, 1H); HRMS (FAB) m/z calcd for C25H36N3O6
[M+H]+: 474.5822, found: 474.5826.
(10 ml) and silver oxide (0.3 g) were added, and the mixture (pro-
tected from light) was stirred for 2–3 h at 45 °C (the reaction was
monitored by TLC). Silver oxide was removed by centrifugation
and the supernatant was evaporated in vacuo to dryness. The res-
idue was dissolved in chloroform (100 ml) and the solution was
washed with 5% aqueous solution of NaOH (30 ml). The aqueous
layer was separated and pH of aqueous solution was brought to
three, and the product was extracted with chloroform. The organic
layer was washed with saturated aqueous NaCl (50 ml). The organ-
ic layer was dried over anhydrous Na2SO4, filtered, and chloroform
was evaporated to yield oily residue of 6. After a period of few days,
oil was converted into solid waxy substance.
4.1.7.3. {6-[4-(1-Boc-piperidin-4-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}acetic acid methyl ester (15C). Mp = 189–
191 °C; 1H NMR d (400 MHz, d6-DMSO) 1.43 (s, 9H), 1.55 (ddd,
J = 24.9, 12.4, 3.8 Hz, 2H), 1.79 (d, J = 12.7 Hz, 2H), 2.75–2.90 (m,
3H), 4.10 (d, J = 10.8 Hz, 2H), 4.29 (s, 2H), 4.50 (s, 2H), 7.44 (d,
J = 7.6 Hz, 2H), 7.59 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 7.3 Hz, 2H), 7.97
(d, J = 8.3 Hz, 1H), 8.22 (s, 1H), 10.41 (s, 1H); HRMS (FAB) m/z calcd
for C28H34N3O6 [M+H]+: 508.5997, found: 508.6002.
4.1.6. 2-Acetyl-1,2,3,4-tetrahydroisoquinoline-7-carboxylic (13)
Compound 12 (2.5 g, 0.117 mol) was dissolved in anhydrous
CH2Cl2 (50 ml). The solution was cooled to À10 °C, and with inten-
sive stirring oxalyl chloride (5 ml, 0.117 mol) was added. AlCl3
(2.85, 0.643 mol) was added in small portions to the stirred solu-
tion with temperature kept below À9 °C. The mixture was stirred
for 1 h at À10 °C. Then additional AlCl3 (3.81, 0.643 mol) was
added, and the mixture was stirred for another 2 h at À10 °C. The
reaction mixture was allowed to stand overnight at room temper-
ature and then poured onto ice (100 g). The aqueous phase was ex-
tracted with CH2Cl2 (100 ml). The organic phase was dried with
anhydrous Na2SO4, filtered, and the solvent was evaporated in va-
cuo. The residue was dissolved in acid, with stirring and heating
(40 °C). To the warm solution, ice was added. pH of the mixture
was brought to three and the product was extracted with chloro-
form. The chloroform phase was dried with anhydrous Na2SO4, fil-
tered, and the solvent was evaporated in vacuo to dryness. The
resulting 13 obtained as a solid residue was used in subsequent
transformations without further purification. Cream powder, yield
53%. Mp = 183.5–184.5 °C.
4.1.7.4. {6-[4-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}acetic acid methyl ester (15d). Mp = 219–
220.5 °C; 1H NMR d (500 MHz, d6-DMSO) 1.44 (s, 9H), 3.31 (t,
J = 4.6 Hz, 4H), 3.48 (t, J = 4.6 Hz, 4H), 3.70 (s, 3H), 4.41 (s, 2H), 4.50
(s, 2H), 7.05 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.2 Hz, 1H), 7.93 (d,
J = 8.7 Hz, 2H), 7.98 (dd, J = 8.2, 1.0 Hz, 1H), 8.23 (d, J = 1.0, 1H),
10.18 (s, 1H); HRMS (FAB) m/z calcd for C27H33N4O6 [M+H]+:
509.5873, found: 509.5880.
4.1.7.5. {6-[(2-Boc-1,2,3,4-tetrahydroisoquinoline-7-carbonyl)-
amino]-1-oxo-1,3-dihydroisoindol-2-yl}acetic acid methyl ester
(15e). Mp = 134–137 °C; 1H NMR d (400 MHz, d6-DMSO) 1.45 (s,
9H), 2.87 (t, J = 5.8 Hz, 2H), 3.60 (t, J = 5.8 Hz, 2H), 3.70 (s, 3H), 4.42
(s, 2H), 4.51 (s, 2H), 4.61 (s, 2H), 7.35 (d, J = 8.1 Hz, 1H), 7.61 (d,
J = 8.3 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.83 (s, 1H), 8.00 (dd, J = 8.2,
1.7 Hz, 1H), 8.24 (d, J = 1.7 Hz, 1H), 10.41 (s, 1H); HRMS (FAB) m/z
calcd for C26H30N3O6 [M+H]+: 480.5455, found: 480.5447.
4.1.7.6. 3-{6-[2-(1-Boc-piperidin-4-yl)acetylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}propionic acid methyl ester (15f).
Mp = 168–170 °C; 1H NMR d (500 MHz, d6-DMSO) 1.09 (ddd,
J = 23.9, 11.7, 3.9 Hz, 2H), 1.40 (s, 9H), 1.67 (d, J = 11.7 Hz, 2H),
1.94–1.98 (m, 1H), 2.28 (d, J = 6.7 Hz, 2H), 2.70 (t, J = 6.7 Hz, 2H),
2.74–2.80 (m, 2H), 3.61 (s, 3H), 3.76 (t, J = 6.7 Hz, 2H), 3.92 (d,
J = 9.1 Hz, 2H), 4.43 (s, 5H), 7.50 (d, J = 8.3 Hz, 1H), 7.68 (d,
J = 8.3 Hz, 1H), 8.04 (s, 1H), 10.12 (s, 1H); HRMS (FAB) m/z calcd
for C24H34N3O6 [M+H]+: 460.5551, found: 460.5560.
4.1.7. General procedure for a preparation of compound 15
The 10 mmol of acid (6–8, 11a,b or 14) was dissolved in anhy-
drous acetonitryle (25 ml). The solution was cooled to À5 °C, and
triethylamine (1.4 ml, 10 mmol), and then HBTU (3.79 g, 10 mmol)
or HATU (3.8 g, 10 mmol), were added. The mixture was stirred for
1 h at À5 °C and then 10 mmol of amine 5 was added. The reaction
mixture was allowed to stand overnight at room temperature. The
residual amount of the activated ether (Bt- or At-ether of starting
acid) was destroyed by addition of few drops of N,N-dimethylpro-
pane-1,3-diamine, and the solvent was evaporated in vacuo to dry-
ness. The residue was dissolved in 100 ml of chloroform. The
solution was washed with water (40 mL), aqueous solution of
1 M HCl (40 ml) and 5% aqueous solution of NaHCO3 (40 ml). The
organic layer was dried over Na2SO4, filtered off, and the solvent
was evaporated in vacuo to dryness. The resulting residue was trit-
urated with warm hexane (20 ml), and the precipitate was col-
lected by filtration and dried.
4.1.7.7. 3-{6-[4-(1-Boc-piperidin-4-yl)butyrylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}propionic
acid
methyl
ester
(15g). Mp = 171–172 °C; 1H NMR d (400 MHz, d6-DMSO) 0.94
(ddd, J = 24.2, 12.2, 4.2 Hz, 2H), 1.23 (dd, J = 15.2, 6.8 Hz, 2H), 1.38
(s, 9H), 1.40–1.45 (m, 1H), 1.58–1.65 (m, 4H), 2.31 (t, J = 7.5 Hz,
2H), 2.58–2.65 (m, 2H), 2.68 (t, J = 7.0 Hz, 2H), 3.60 (s, 3H), 3.74 (t,
J = 7.0 Hz, 2H), 3.91 (d, J = 12.5 Hz, 2H), 4.41 (s, 2H), 7.49 (d,
J = 8.2 Hz, 1H), 7.67 (dd, J = 8.2, 1.7 Hz, 1H), 8.02 (d, J = 1.7 Hz, 1H),
10.08 (s, 1H); HRMS (FAB) m/z calcd for C26H38N3O6 [M+H]+:
488.6093, found: 488.6087.
4.1.7.1. {6-[2-(1-Boc-piperidin-4-yl)acetylamino]-1-oxo-1,3-dihyd-
roisoindol-2-yl}acetic acid methyl ester (15a). Mp = 193–
195 °C; 1H NMR d (400 MHz, d6-DMSO) 1.07 (ddd, J = 24.3, 12.1,
3.9 Hz, 2H), 1.38 (s, 9H), 1.65 (d, J = 11.0 Hz, 2H), 1.94 (m,
J = 3.7 Hz, 1H), 2.27 (d, J = 6.8 Hz, 2H), 2.64–286 (m, 2H), 3.67 (s,
3H), 3.90 (d, J = 11.7 Hz, 2H), 4.37 (s, 2H), 4.45 (s, 2H), 7.52 (d,
J = 8.1 Hz, 1H), 7.68 (dd, J = 8.3, 2.0 Hz, 1H), 8.07 (d, J = 1.2 Hz, 1H),
10.13 (s, 1H); HRMS (FAB) m/z calcd for C23H33N3O6 [M+H]+:
446.5280, found: 446.5273.
4.1.7.8. 3-{6-[4-(1-Boc-piperidin-4-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}propionic
acid
methyl
ester
(15h). Mp = 199.5–200.5 °C; 1H NMR d (400 MHz, d6-DMSO)
1.41 (s, 9H), 1.52 (ddd, J = 25.2, 12.6, 4.0 Hz, 2H), 1.77 (d,
J = 12.6 Hz, 2H), 2.69 (t, J = 7.0 Hz, 2H), 2.73–2.90 (m, 3H), 3.59 (s,
3H), 3.75 (t, J = 7.0 Hz, 2H), 4.08 (d, J = 11.5 Hz, 2H), 4.44 (s, 2H),
7.41 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.1 Hz,
2H), 7.92 (dd, J = 8.3, 1.7 Hz, 1H), 8.16 (d, J = 1.7 Hz, 1H), 10.36 (s,
1H); HRMS (FAB) m/z calcd for C29H36N3O6 [M+H]+: 522.6268,
found: 522.6260.
4.1.7.2.
{6-[4-(1-Boc-piperidin-4-yl)butyrylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}acetic acid methyl ester (15b). Mp = 149–
150 °C; 1H NMR d (400 MHz, d6-DMSO) 0.95 (ddd, J = 24.3, 12.2,

A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
4655
4.1.7.9. 3-{6-[4-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}propionic acid methyl ester (15i). Mp = 185–
187 °C; 1H NMR d (400 MHz, d6-DMSO) 1.43 (s, 9H), 2.71 (t, J = 6.9 Hz,
2H), 3.30 (s, 4H), 3.48 (s, 4H), 3.61 (s, 3H), 3.77 (t, J = 6.9 Hz, 2H), 4.45 (s,
2H), 7.04 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 8.7 Hz, 1H), 7.91 (d, J = 9.0 Hz,
2H), 7.95 (dd, J = 8.7, 1.7 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 10.15 (s,
1H); HRMS (FAB) m/z calcd for C28H35N4O6 [M+H]+: 523.6144, found:
523.6137.
7.95 (dd, J = 8.2, 1.8 Hz, 1H), 8.17 (d, J = 1.8 Hz, 1H), 10.15 (s, 1H);
HRMS (FAB) m/z calcd for C29H37N4O6 [M+H]+: 537.6414, found:
537.66417.
4.1.7.16. 3-{6-[(2-Boc-1,2,3,4-tetrahydroisoquinoline-7-carbonyl)-
amino]-1-oxo-1,3-dihydroisoindol-2-yl}butyric acid methyl
ester (15p).
1H NMR d (400 MHz, d6-DMSO + CCl4) 1.29 (d,
J = 6.6 Hz, 3H), 1.45 (s, 9H), 2.67–2.77 (m, 2H), 2.86 (t, J = 5.0 Hz,
2H), 3.57 (s, 3H), 3.58–3.60 (m, 2H), 4.40 (dd, J = 22.0, 17.4 Hz,
2H), 4.59–4.66 (m, 3H), 7.33 (d, J = 7.5 Hz, 1H), 7.57 (d, J = 8.3 Hz,
1H), 7.79–7.82 (m, 2H), 7.95 (d, J = 7.9 Hz, 1H), 8.18 (s, 1H), 10.36
(s, 1H); HRMS (FAB) m/z calcd for C28H34N3O6 [M+H]+: 508.5997,
found: 508.5995.
4.1.7.10. 3-{6-[(2-Boc-1,2,3,4-tetrahydroisoquinoline-7-carbonyl)
amino]-1-oxo-1,3-dihydroisoindol-2-yl}propionic acid methyl
ester (15j).
Mp = 102–103 °C; 1H NMR d (400 MHz, d6-DMSO)
1.45 (s, 9H), 2.72 (t, J = 7.5 Hz, 2H), 2.87 (t, J = 5.9 Hz, 2H), 3.60 (t,
J = 5.6 Hz, 2H), 3.62 (s, 3H), 3.78 (t, J = 7.0 Hz, 2H), 4.47 (s, 2H),
4.61 (s, 2H), 7.34 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H), 7.80
(d, J = 8.1 Hz, 1H), 7.82 (s, 1H), 7.95 (dd, J = 8.1, 1.5 Hz, 1H), 8.19
(d, J = 1.5 Hz, 1H), 10.37 (s, 1H); HRMS (FAB) m/z calcd for
4.1.7.17. 3-{6-[3-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}butyric acid methyl ester (15q). Mp = 142–
143.5 °C; 1H NMR d (400 MHz, d6-DMSO) 1.30 (d, J = 6.7 Hz, 3H),
1.44 (s, 9H), 2.68–2.79 (m, 2H), 3.24 (s, 4H), 3.53 (s, 4H), 3.57 (s,
3H), 4.43 (dd, J = 24.4, 17.1 Hz, 2H), 4.63 (m, 1H), 7.25 (d, J = 7.8 Hz,
1H), 7.41 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.58 (d,
J = 7.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 1H), 8.17 (s, 1H), 10.40 (s, 1H);
HRMS (FAB) m/z calcd for C29H37N4O6 [M+H]+: 537.6414, found:
537.66416.
C
27H32N3O6 [M+H]+: 494.5726, found: 494.5730.
4.1.7.11. 3-{6-[3-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-
1,3-dihydroisoindol-2-yl}propionic acid methyl ester
(15k). (500 MHz, d6-DMSO)
Mp = 181–183 °C; 1H NMR
d
1.44 (s, 9H), 2.72 (t, J = 6.5 Hz, 2H), 3.21 (s, 4H), 3.50 (s, 4H), 3.62
(s, 3H), 3.78 (t, J = 6.1 Hz, 2H), 4.47 (s, 2H), 7.19 (d, J = 6.2 Hz,
1H), 7.38–7.44 (m, 2H), 7.50 (s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.95
(d, J = 8.0 Hz, 1H), 8.17 (m, 1H), 10.35 (s, 1H); HRMS (FAB) m/z
calcd for C28H35N4O6 [M+H]+: 523.6144, found: 523.6146.
4.1.7.18. 4-{6-[2-(1-Boc-piperidin-4-yl)acetylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}butyric acid methyl ester (15r). Mp = 159–
160 °C; 1H NMR d (500 MHz, d6-DMSO) 1.06–1.12 (m, 2H), 1.40 (s,
9H), 1.67 (d, J = 11.7 Hz, 2H), 1.87 (t, J = 6.8, 2H), 1.92–1.99 (m, 1H),
2.28 (d, J = 6.5, 2H), 2.34 (t, J = 6.8, 2H), 2.65–2.80 (m, 2H), 3.52–
3.56 (m, 5H), 3.92 (d, J = 8.6 Hz, 2H), 4.40 (s, 2H), 7.50 (d, J = 7.9 Hz,
1H), 7.68 (d, J = 7.9 Hz, 1H), 8.03 (s, 1H), 10.09 (s, 1H); HRMS (FAB)
m/z calcd for C25H36N3O6 [M+H]+: 474.5822, found: 474.5819.
4.1.7.12. 3-{6-[2-(1-Boc-piperidin-4-yl)acetylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}butyric acid methyl ester (15l). Mp = 130–
132 °C; 1H NMR d (500 MHz, d6-DMSO) 1.09 (ddd, J = 22.5, 11.7,
3.1 Hz, 2H), 1.28 (d, J = 5.7 Hz, 3H), 1.40 (s, 9H), 1.67 (d, J = 11.7 Hz,
2H), 1.91–2.00 (m, 1H), 2.27 (d, J = 6.2 Hz, 2H), 2.65–2.82 (m, 4H),
3.56 (s, 3H), 3.93 (d, J = 8.3 Hz, 2H), 4.38 (dd, J = 22.8, 17.1 Hz, 2H),
4.60 (m, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 8.03 (s,
1H), 10.10 (s, 1H); HRMS (FAB) m/z calcd for C25H36N3O6 [M+H]+:
474.5822, found: 474.5823.
4.1.7.19. 4-{6-[4-(1-Boc-piperidin-4-yl)butyrylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}butyric
(15s).
acid
methyl
(400 MHz, d6-DMSO)
ester
Mp = 151–153 °C; 1H NMR
d
0.94 (ddd, J = 24.4, 12.2, 4.0 Hz, 2H), 1.23 (dd, J = 15.7, 7.3 Hz,
2H), 1.38 (s, 9H), 1.41–1.43 (m, 1H), 1.60–1.66 (m, 4H), 1.86 (dt,
J = 14.2, 7.1 Hz, 2H), 2.32 (m, J = 7.1 Hz, 4H), 2.59–2.73 (m, 2H),
3.52 (t, J = 7.1 Hz, 2H), 3.53 (s, 3H), 3.91 (d, J = 11.7 Hz, 2H), 4.39
(s, 2H), 7.49 (d, J = 8.1 Hz, 1H), 7.68 (dd, J = 8.1, 1.6 Hz, 1H), 8.2
(d, J = 1.6 Hz, 1H), 10.07 (s, 1H); HRMS (FAB) m/z calcd for
4.1.7.13.
1,3-dihydroisoindol-2-yl}butyric
3-{6-[4-(1-Boc-piperidin-4-yl)butyrylamino]-1-oxo-
acid methyl ester
(15m). Mp = 133–134 °C; 1H NMR d (500 MHz, d6-DMSO) 0.95
(ddd, J = 24.0, 12.2, 3.9 Hz, 2H), 1.22–1.25 (m, 2H), 1.28 (d,
J = 6.6 Hz, 3H), 1.39 (s, 9H), 1.61–1.66 (m, 4H), 2.32 (t, J = 7.3 Hz,
2H), 2.65–2.77 (m, 4H), 3.56 (s, 3H), 3.92 (d, J = 9.3 Hz, 2H), 4.38
(dd, J = 24.0, 17.1 Hz, 2H), 4.61 (td, J = 13.7, 6.8 Hz, 1H), 7.50 (d,
J = 8.3 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 8.02 (s, 1H), 10.07 (s, 1H);
HRMS (FAB) m/z calcd for C27H40N3O6 [M+H]+: 502.6364, found:
502.6367.
C
27H40N3O6 [M+H]+: 502.6364, found: 502.6363.
4.1.7.20. 4-{6-[4-(1-Boc-piperidin-4-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}butyric acid methyl ester (15t). Mp = 166–
167 °C; 1H NMR d (400 MHz, d6-DMSO) 1.4 (s, 9H) 1.5 (ddd,
J = 22.3, 12.0, 2.4 Hz, 2H) 1.8 (d, J = 12.2 Hz, 2H) 1.9 (dt, J = 13.4,
6.6 Hz, 2H) 2.3 (t, J = 6.6 Hz, 2H) 2.8 (m, 3H) 3.5 (m, 5H) 4.1 (d,
J = 9.0 Hz, 2H) 4.4 (s, 2H) 7.4 (d, J = 7.3 Hz, 2H) 7.5 (d, J = 8.1 Hz, 1H)
7.9 (m, J = 6.7, 6.7 Hz, 3H) 8.2 (s, 1H) 10.4 (s, 1H); HRMS (FAB) m/z
calcd for C30H38N3O6 [M+H]+: 536.6539, found: 536.6537.
4.1.7.14. 3-{6-[4-(1-Boc-piperidin-4-yl)benzoylamino]-1-oxo-
1,3-dihydroisoindol-2-yl}butyric acid methyl ester (15n). 1H
NMR d (500 MHz, d6-DMSO) 1.30 (d, J = 6.8 Hz, 3H), 1.43 (s, 9H),
1.55 (ddd, J = 24.5, 11.7, 2.9 Hz, 2H), 1.79 (d, J = 11.7 Hz, 2H), 2.68–
2.85 (m, 5H), 3.57 (s, 3H), 4.11 (d, J = 7.6 Hz, 2H), 4.42 (dd, J = 24.0,
17.6 Hz, 2H), 4.63 (dt, J = 13.9, 6.7 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H),
7.57 (d, J = 8.1 Hz, 1H), 7.92–7.96 (m, 3H), 8.18 (s, 1H), 10.38 (s,
1H); HRMS (FAB) m/z calcd for C30H38N3O6 [M+H]+: 536.6539,
found: 536.6535
4.1.7.21. 4-{6-[(2-Boc-1,2,3,4-tetrahydroisoquinoline-7-carbonyl)-
amino]-1-oxo-1,3-dihydroisoindol-2-yl}butyric acid methyl
ester (15u).
Mp = 142–145 °C; 1H NMR
d (400 MHz, d6-
DMSO) 1.44 (s, 9H), 1.88 (dt, J = 13.8, 6.6 Hz, 4H), 2.35 (t,
J = 7.1 Hz, 2H), 2.86 (t, J = 4.9, 2H), 3.54 (s, 3H), 3.59 (t, J = 4.9,
4H), 4.44 (s, 2H), 4.60 (s, 2H), 7.33 (d, J = 7.6 Hz, 1H), 7.56 (d,
J = 8.1 Hz, 2H), 7.78–7.81 (m, 2H), 7.94 (d, J = 8.1 Hz, 1H), 8.18 (s,
1H), 10.37 (s, 1H); HRMS (FAB) m/z calcd for C28H34N3O6 [M+H]+:
508.5997, found: 508.5995.
4.1.7.15. 3-{6-[4-(1-Boc-piperazin-4-yl)benzoylamino]-1-oxo-
1,3-dihydroisoindol-2-yl}butyric
acid
methyl
ester
(15o). Mp = 95–97 °C (decomposes); 1H NMR d (500 MHz, d6-
DMSO) 1.29 (d, J = 6.8 Hz, 3H), 1.44 (s, 9H), 2.68–2.78 (m, 2H), 3.30
(t, J = 4.0 Hz, 4H), 3.48 (t, J = 4.0 Hz, 4H), 3.57 (s, 3H), 4.41 (dd,
J = 24.2, 17.4 Hz, 2H), 4.63 (td, J = 13.9, 6.5 Hz, 1H), 7.05 (d,
J = 9.0 Hz, 2H), 7.55 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 9.0 Hz, 2H),
4.1.8. General procedure for a preparation of compound 16
The compound 15 (10 mmol) was dissolved in methanol
(25 ml), and to this solution was added 1 M NaOH aqueous solu-

4656
A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
tion (5 ml). The reaction mixture was left at room temperature
overnight. Then chloroform (50 ml) was added, and pH of the mix-
ture was brought to three with intensive stirring. When the prod-
uct was not soluble in chloroform layer, an suspension was formed,
the product was filtered off. The precipitate was washed (on the fil-
ter) with water, chloroform and ether, and dried in air. Thus ob-
tained product 16 was not need to be further purified. When the
reaction product was soluble in chloroform, the chloroform phase
was collected and washed with water (20 ml). The chloroform
phase was dried with anhydrous Na2SO4, filtered, and the solvent
was evaporated in vacuo to dryness. The resulting residue was trit-
urated with ether (20 ml) and the precipitate was collected by fil-
tration and dried in air.
2H), 3.72 (t, J = 7.1 Hz, 2H), 3.92 (d, J = 11.7 Hz, 2H), 4.43 (s, 2H),
7.51 (d, J = 8.1 Hz, 1H), 7.67 (dd, J = 8.1, 1.2 Hz, 1H), 8.04 (d,
J = 1.2 Hz, 1H), 10.11 (s, 1H), 12.36 (br s, 1H); HRMS (FAB) m/z calcd
for C23H32N3O6 [M+H]+: 446.5280, found: 546.5274.
4.1.8.7. 3-{6-[4-(1-Boc-piperidin-4-yl)butyrylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}propionic acid (16g).
Mp = 176.5–
177.5 °C; 1H NMR d (400 MHz, d6-DMSO) 0.93 (ddd, J = 23.0, 10.5,
3.2 Hz, 2H), 1.22 (dd, J = 14.2, 6.9 Hz, 2H), 1.36 (s, 9H), 1.49–1.46
(m, 1H), 1.57–1.64 (m, 4H), 2.30 (t, J = 7.3 Hz, 2H), 2.58 (t,
J = 6.9 Hz, 2H), 2.62–2.76 (m, 2H), 3.69 (t, J = 6.9 Hz, 2H), 3.90 (d,
J = 10.5 Hz, 2H), 4.41 (s, 2H), 7.48 (d, J = 8.1 Hz, 1H), 7.66 (dd,
J = 8.1, 1.5 Hz, 1H), 8.01 (d, J = 1.5 Hz, 1H), 10.10 (s, 1H), 12.34 (br
s, 1H); HRMS (FAB) m/z calcd for C25H36N3O6 [M+H]+: 474.5822,
found: 508.5828.
4.1.8.1. {6-[2-(1-Boc-piperidin-4-yl)acetylamino]-1-oxo-1,3-dihyd-
roisoindol-2-yl}acetic acid (16a). Mp = 198–201 °C; 1H NMR d
(400 MHz, d6-DMSO) 1.07 (ddd, J = 23.6, 11.9, 3.2 Hz, 2H), 1.38 (s,
9H), 1.65 (d, J = 12.2 Hz, 2H), 1.91–197 (m, 1H), 2.26 (d, J = 6.8 Hz,
2H), 2.65–2.80 (m, 2H), 3.91 (d, J = 11.0 Hz, 2H), 4.25 (s, 2H), 4.44 (s,
2H), 7.51 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 8.06 (s, 1H),
10.13 (s, 1H), 12.86 (br s, 1H); HRMS (FAB) m/z calcd for
4.1.8.8. 3-{6-[4-(1-Boc-piperidin-4-yl)benzoylamino]-1-oxo-1,3-
1
dihydroisoindol-2-yl}propionic acid (16h). Mp = 195–196 °C; H
NMR d (400 MHz, d6-DMSO) 1.41 (s, 9H), 1.52 (ddd, J = 25.1,
12.6, 3.9 Hz, 2H), 1.77 (d, J = 12.5 Hz, 2H), 2.60 (t, J = 7.1 Hz,
2H), 2.74–2.85 (m, 3H), 3.72 (t, J = 7.0 Hz, 2H), 4.08 (d, J = 10.3 Hz,
2H), 4.45 (s, 2H), 7.41 (d, J = 8.3 Hz, 2H), 7.54 (d,
J = 8.1 Hz, 1H), 7.90 (d, J = 8.3 Hz, 2H), 7.92 (dd, J = 8.1, 1.7 Hz, 1H),
8.17 (d, J = 1.7 Hz, 1H), 10.37 (s, 1H), 12.37 (br s, 1H); HRMS
C
22H30N3O6 [M+H]+: 432.5009, found: 432.5005.
4.1.8.2.
{6-[4-(1-Boc-piperidin-4-yl)butyrylamino]-1-oxo-1,3-
Mp = 165.5–167 °C;
dihydroisoindol-2-yl}acetic acid (16b).
(FAB) m/z calcd for
C
28H34N3O6 [M+H]+: 508.5997, found:
1H NMR d (400 MHz, d6-DMSO) 0.95 (ddd, J = 24.3, 12.3, 4.0 Hz,
2H), 1.22–1.27 (m, 2H), 1.39 (s, 9H), 1.42–1.45 (m, 1H), 1.59–168
(m, 4H), 2.33 (t, J = 7.5 Hz, 2H), 2.60–2.75 (m, 2H), 3.93 (d,
J = 11.2 Hz, 2H), 4.27 (s, 2H), 4.46 (s, 2H), 7.53 (d, J = 8.1 Hz, 1H),
7.71 (dd, J = 8.3, 1.7 Hz, 1H), 8.08 (d, J = 1.5 Hz, 1H), 10.12 (s, 1H),
12.96 (br s, 1H); HRMS (FAB) m/z calcd for C24H32N3O6 [M+H]+:
460.5551, found: 460.5559.
508.6002.
4.1.8.9. 3-{6-[4-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}propionic acid (16i). Mp >300 °C (decom-
poses); 1H NMR
d (400 MHz, d6-DMSO) 1.43 (s, 9H), 2.62 (t,
J = 6.0 Hz, 2H), 3.30 (s, 4H), 3.48 (s, 4H), 3.74 (t, J = 6.0 Hz, 2H), 4.46
(s, 2H), 7.04 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 7.5 Hz, 1H), 7.91–7.95 (m,
2H), 8.18 (s, 1H), 10.15 (s, 1H), 12.35 (br s, 1H); HRMS (FAB) m/z calcd
for C27H33N4O6 [M+H]+: 509.5873, found: 509.5870.
4.1.8.3. {6-[4-(1-Boc-piperidin-4-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}acetic acid (16C).
Mp = 223–225 °C;
1H NMR d (400 MHz, d6-DMSO) 1.43 (s, 9H), 1.53 (ddd, J = 24.9,
12.4, 3.8 Hz, 2H), 1.79 (d, J = 12.7 Hz, 2H), 2.77–2.85 (m, 3H), 4.11
(d, J = 10.8 Hz, 2H), 4.30 (s, 2H), 4.50 (s, 2H), 7.44 (d, J = 7.6 Hz,
2H), 7.59 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 7.3 Hz, 2H), 7.97 (d,
J = 8.3 Hz, 1H), 8.27 (s, 1H), 10.41 (s, 1H); HRMS (FAB) m/z calcd
for C27H32N3O6 [M+H]+: 494.5726, found: 494.5721.
4.1.8.10.
3-{6-[(2-Boc-1,2,3,4-tetrahydroisoquinoline-7-carbonyl)
acid (16j)
amino]-1-oxo-1,3-dihydroisoindol-2-yl}propionic
Mp = 118–120 °C (decomposes); 1H NMR d (400 MHz, d6-DMSO) 1.45
(s, 9H), 2.63 (t, J = 7.1 Hz, 2H), 2.87 (t, J = 5.7 Hz, 2H), 3.60 (t,
J = 5.5 Hz, 2H), 3.75 (t, J = 7.0 Hz, 2H), 4.48 (s, 2H), 4.61 (s, 2H), 7.34
(d, J = 8.1 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.83
(s, 1H), 7.95 (dd, J = 8.2, 1.6 Hz, 1H), 8.19 (d, J = 1.6 Hz, 1H), 10.38 (s,
1H), 12.35 (br s, 1H); HRMS (FAB) m/z calcd for C26H30N3O6 [M+H]+:
480.5455, found: 480.5457.
4.1.8.4. {6-[4-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}acetic acid (16d).
Mp = 215–216.5 °C
(decomposes); 1H NMR d (500 MHz, d6-DMSO) 1.44 (s, 9H), 3.31
(s, 4H), 3.48 (s, 4H), 4.29 (s, 2H), 4.49 (s, 2H), 7.05 (d, J = 8.3 Hz,
2H), 7.56 (d, J = 8.3 Hz, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.98 (d,
J = 8.3 Hz, 1H), 8.23 (s, 1H), 10.17 (s, 1H), 12.97 (br s, 1H); HRMS
4.1.8.11. 3-{6-[3-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-
1,3-dihydroisoindol-2-yl}propionic acid (16k). Mp = 196–
196.5 °C; 1H NMR d (500 MHz, d6-DMSO) 1.43 (s, 9H), 2.53–
2.54 (m, 2H), 3.21 (t, J = 6.4 Hz, 4H), 3.49 (t, J = 6.4 Hz, 4H),
3.72 (t, J = 6.8 Hz, 2H), 4.48 (s, 2H), 7.18 (d, J = 7.6 Hz, 1H),
7.38 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.53–7.56 (m,
2H), 7.98 (d, J = 8.1 Hz, 1H), 8.19 (s, 1H), 10.49 (s, 1H); HRMS
(FAB) m/z calcd for
C
26H31N4O6 [M+H]+: 495.5602, found:
495.5610.
4.1.8.5. {6-[(2-Boc-1,2,3,4-tetrahydroisoquinoline-7-carbonyl)amino]-
1-oxo-1,3-dihydroisoindol-2-yl}acetic acid (16e). Mp = 218–
(FAB) m/z calcd for
C
27H33N4O6 [M+H]+: 509.5873, found:
220 °C (decomposes); 1H NMR d (400 MHz, d6-DMSO) 1.45 (s,
9H), 2.87 (t, J = 5.7 Hz, 2H), 3.60 (t, J = 5.7 Hz, 2H), 4.29 (s, 2H),
4.50 (s, 2H), 4.61 (s, 2H), 7.35 (d, J = 8.1 Hz, 1H), 7.60 (d,
J = 8.3 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.83 (s, 1H), 7.98 (dd,
J = 8.3, 2.0 Hz, 1H), 8.23 (d, J = 1.7 Hz, 1H), 10.41 (s, 1H), 12.96 (br
s, 1H); HRMS (FAB) m/z calcd for C25H28N3O6 [M+H]+: 466.5184,
found: 466.5187.
509.5879.
4.1.8.12. 3-{6-[2-(1-Boc-piperidin-4-yl)acetylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}butyric acid (16l). Mp = 197–199 °C;
1H NMR d (500 MHz, d6-DMSO) 1.09 (ddd, J = 23.6, 11.9, 3.1 Hz,
2H), 1.27 (d, J = 6.5 Hz, 3H), 1.40 (s, 9H), 1.67 (d, J = 12.2 Hz, 2H),
1.91–2.02 (m, 1H), 2.28 (d, J = 7.3 Hz, 2H), 2.62 (ddd, J = 31.5,
15.2, 7.3 Hz, 2H), 2.69–2.80 (m, 2H), 3.92 (d, J = 9.1 Hz, 2H), 4.39
(dd, J = 27.5, 17.4 Hz, 2H), 4.60 (td, J = 13.1, 6.0 Hz, 1H), 7.51 (d,
J = 8.3 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 8.02 (s, 1H), 10.09 (s, 1H),
12.26 (s, 1H); HRMS (FAB) m/z calcd for C24H34N3O6 [M+H]+:
460.5551, found: 450.5555.
4.1.8.6. 3-{6-[2-(1-Boc-piperidin-4-yl)acetylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}propionic
acid
(16f).
Mp = 193–
194 °C; 1H NMR d (400 MHz, d6-DMSO) 1.08 (ddd, J = 24.6, 12.4,
3.3 Hz, 2H), 1.40 (s, 9H), 1.66 (d, J = 11.5 Hz, 2H), 1.90–2.01 (m,
1H), 2.28 (d, J = 7.1 Hz, 2H), 2.60 (t, J = 7.0 Hz, 2H), 2.66–2.82 (m,

A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
4657
4.1.8.13.
1,3-dihydroisoindol-2-yl}butyric
3-{6-[4-(1-Boc-piperidin-4-yl)butyrylamino]-1-oxo-
acid (16m). Mp = 139–
HRMS (FAB) m/z calcd for C26H37N3O6 [M+H]+: 488.6093, found:
488.6090.
140.5 °C; 1H NMR d (500 MHz, d6-DMSO) 0.95 (ddd, J = 24.5, 12.2,
3.7 Hz, 2H), 1.20–1.25 (m, 5H), 1.39 (s, 9H), 1.42–1.44 (m, 1H),
1.59–1.65 (m, 4H), 2.32 (t, J = 7.1 Hz, 2H), 2.62–2.69 (m, 2H), 3.92
(d, J = 8.1 Hz, 2H), 4.36 (s, 2H), 4.59 (m, 1H), 7.46 (d, J = 7.8 Hz, 1H),
7.69 (d, J = 7.8 Hz, 1H), 8.03 (s, 1H), 10.23 (s, 1H); HRMS (FAB) m/z
calcd for C26H38N3O6 [M+H]+: 488.6093, found: 488.6089.
4.1.8.20. 4-{6-[4-(1-Boc-piperidin-4-yl)benzoylamino]-1-oxo-
1,3-dihydroisoindol-2-yl}butyric acid (16t).
Mp = 197–
197.5 °C; 1H NMR d (400 MHz, d6-DMSO) 1.42 (s, 9H), 1.53 (ddd,
J = 25.1, 12.8, 3.9 Hz, 2H), 1.78 (d, J = 13.7 Hz, 2H), 1.84 (dt,
J = 14.2, 6.8 Hz, 2H), 2.25 (t, J = 7.2 Hz, 2H), 2.75–2.85 (m, 3H),
3.54 (t, J = 6.8 Hz, 2H), 4.09 (d, J = 9.8 Hz, 2H), 4.44 (s, 2H), 7.42
(d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.3 Hz, 1H), 7.91–7.94 (m, 3H), 8.17
(s, 1H), 10.38 (s, 1H); HRMS (FAB) m/z calcd for C29H36N3O6
[M+H]+: 522.6268, found: 522.6261.
4.1.8.14. 3-{6-[4-(1-Boc-piperidin-4-yl)benzoylamino]-1-oxo-
1,3-dihydroisoindol-2-yl}butyric acid (16n). Mp = 175–176 °C;
1H NMR d (500 MHz, d6-DMSO) 1.25 (d, J = 6.4 Hz, 3H), 1.43 (s, 9H),
1.54 (ddd, J = 24.8, 12.2, 3.5 Hz, 2H), 1.79 (d, J = 12.2 Hz, 2H), 2.74–
2.89 (m, 3H), 4.10 (d, J = 6.4 Hz, 2H), 4.41 (s, 2H), 4.62 (dt, J = 13.5,
6.5 Hz, 1H), 7.42 (d, J = 7.8 Hz, 2H), 7.55 (d, J = 8.3 Hz, 1H), 7.93–
7.96 (m, 3H), 8.18(s, 1H), 10.43 (s, 1H); HRMS (FAB) m/z calcd for
4.1.8.21. 4-{6-[(2-Boc-1,2,3,4-tetrahydroisoquinoline-7-carbonyl)-
amino]-1-oxo-1,3-dihydroisoindol-2-yl}butyric
(16u).
acid
Mp = 90–93 °C; 1H NMR d (400 MHz, d6-DMSO) 1.44
C
29H36N3O6 [M+H]+: 522.6268, found: 522.6270.
(s, 9H), 1.84 (dt, J = 13.9, 7.1 Hz, 2H), 2.26 (t, J = 7.0 Hz, 2H), 2.86
(t, J = 5.1 Hz, 2H), 3.54 (t, J = 6.7 Hz, 2H), 3.59 (t, J = 4.6 Hz, 2H),
4.45 (s, 2H), 4.60 (s, 2H), 7.33 (d, J = 7.6 Hz, 1H), 7.56 (d,
J = 8.1 Hz, 2H), 7.78–7.82 (m, 2H), 7.94 (d, J = 8.1 Hz, 1H), 8.18 (s,
1H), 10.37 (s, 1H), 12.14 (br s, 1H); HRMS (FAB) m/z calcd for
4.1.8.15. 3-{6-[4-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-
1,3-dihydroisoindol-2-yl}butyric acid (16o). Mp = 155.5–
156 °C; 1H NMR d (500 MHz, d6-DMSO) 1.29 (d, J = 6.6 Hz, 3H),
1.44 (s, 9H), 2.64 (ddd, J = 32.3, 15.3, 7.5 Hz, 2H), 3.30 (s, 4H), 3.48
(s, 4H), 4.41 (dd, J = 27.1, 17.1 Hz, 2H), 4.62 (td, J = 13.8, 7.0 Hz, 1H),
7.04 (d, J = 8.8 Hz, 2H), 7.55 (d, J = 8.1 Hz, 1H), 7.93 (d, J = 8.1 Hz,
2H), 7.95 (d, J = 8.8 Hz, 1H), 8.18 (s, 1H), 10.16 (s, 1H), 12.27 (br s,
1H); HRMS (FAB) m/z calcd for C28H35N4O6 [M+H]+: 523.6144,
found: 523.6139.
C
27H32N3O6 [M+H]+: 494.5726, found: 494.5721.
4.1.9. General procedures for a preparation of compound 17
Method A. This method was used when the compound 16 was
not soluble in anhydrous CH2Cl2. The compound 16 (10 mmol)
was suspended in anhydrous CH2Cl2, and trifluoroacetic acid
(1 ml) was added. The precipitate was dissolved in a short time,
and the reaction solution was kept for 2 h, protected from mois-
ture. After the solvent was evaporated in vacuo to dryness, the res-
idue was dried in vacuo (2 mmHg) for 2 h at 40 °C.
Method B. This method was used for the compound 16 soluble in
anhydrous CH2Cl2. The compound 16 (10 mmol) was dissolved in
anhydrous CH2Cl2, and the stream of dry HCl was passed through
the solution for 1 h. The solvent was evaporated, and the solid res-
idue was dried in vacuo (2 mm Hg) for 2 h at 40 °C.
4.1.8.16. 3-{6-[(2-Boc-1,2,3,4-tetrahydroisoquinoline-7-carbonyl)-
amino]-1-oxo-1,3-dihydroisoindol-2-yl}-butyric
acid
(16p). Mp = 153–154 °C; 1H NMR d (500 MHz, d6-DMSO) 1.29
(d, J = 6.6 Hz, 3H), 1.45 (s, 9H), 2.64 (ddd, J = 32.8, 15.2, 7.5 Hz, 2H),
2.87 (t, J = 5.2 Hz, 2H), 3.60 (t, J = 5.2 Hz, 2H), 4.43 (dd, J = 27.1,
17.4 Hz, 2H), 4.55–4.64 (m, 3H), 7.34 (d, J = 7.8 Hz, 1H), 7.57
(d, J = 8.3 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.83 (s, 1H), 7.95 (dd,
J = 8.1, 1.5 Hz, 2H), 8.18 (s, 1H) 10.36 (s, 1H), 12.30 (br s, 1H);
HRMS (FAB) m/z calcd for C27H32N3O6 [M+H]+: 494.5726, found:
493.5723.
4.1.9.1. [1-Oxo-6-(2-piperidinium-4-yl-acetylamino)-1,3-dihyd-
roisoindol-2-yl]acetic acid trifluoroacetate (17a). Mp = 236–
239 °C; 1H NMR d (500 MHz, d6-DMSO) 1.41 (ddd, J = 22.6, 10.6,
2.6 Hz, 2H), 1.85 (d, J = 13.8 Hz, 2H), 2.05–2.13 (m, 1H), 2.33 (d,
J = 6.7 Hz, 2H), 2.91 (dd, J = 22.8, 11.6 Hz, 2H), 3.27 (d, J = 11.9 Hz,
2H), 4.28 (s, 2H), 4.46 (s, 2H), 7.54 (d, J = 8.2 Hz, 1H), 7.69 (d,
J = 8.2 Hz, 1H), 8.10 (s, 1H), 8.37 (d, J = 8.7 Hz, 1H), 8.65 (d, J = 8.7 Hz,
1H), 10.23 (s, 1H); 13C NMR d (125 MHz, d6-DMSO) 28.64 (2C), 31.17
(1C), 43.05 (1C), 43.58 (2C), 43.95 (1C), 50.41 (1C), 113.61 (1C),
4.1.8.17. 3-{6-[3-(4-Boc-piperazin-1-yl)benzoylamino]-1-oxo-
1,3-dihydroisoindol-2-yl}butyric acid (16q). Mp >300 °C; 1H
NMR d (500 MHz, d6-DMSO) 1.29 (d, J = 3.9 Hz, 3H), 1.44 (s, 9H),
2.65 (ddd, J = 33.2, 18.0, 6.4 Hz, 2H), 3.22 (s, 4H), 3.51 (s, 4H), 4.43
(dd, J = 26.2, 16.1 Hz, 2H), 4.62 (td, J = 14.1, 6.2 Hz, 1H), 7.20 (d,
J = 4.4 Hz, 1H), 7.38–7.43 (m, 2H), 7.51 (s, 1H), 7.58 (d, J = 6.5 Hz,
1H), 7.95 (d, J = 7.0 Hz, 1H), 8.16 (s, 1H), 10.35 (s, 1H), 12.30 (br s,
1H); HRMS (FAB) m/z calcd for C28H35N4O6 [M+H]+: 523.6144,
found: 523.6139.
116.75 (1C, CF3COÀ), 123.10 (1C), 124.17 (s, 1C), 132.64 (1C), 137.06
2
(1C), 139.42 (1C), 158.81 (1C, CF3COÀ), 168.15 (1C), 170.37 (1C),
2
170.98 (1C); HRMS (FAB) m/z calcd for C17H22N3O4 [M+H]+:
4.1.8.18. 4-{6-[2-(1-Boc-piperidin-4-yl)acetylamino]-1-oxo-1,3-
332.3826, found: 332.3825.
dihydroisoindol-2-yl}butyric acid (16r).
Mp = 191.5–192 °C;
1H NMR d (500 MHz, d6-DMSO) 1.06–1.13 (m, 2H), 1.40 (s, 9H), 1.67
(d, J = 12.2 Hz, 2H), 1.84 (t, J = 7.2, 2H), 1.94–1.99 (m, 1H), 2.25–2.28
(m, 4H), 2.67–2.79(m, 2H), 3.54 (t, J = 7.2 Hz, 2H), 3.93(d, J = 10.1 Hz,
2H), 4.41 (s, 2H), 7.50 (d, J = 7.0 Hz, 1H), 7.68 (d, J = 7.0 Hz, 1H), 8.03
(s, 1H), 10.09 (s, 1H), 12.07 (s, 1H); HRMS (FAB) m/z calcd for
4.1.9.2. [1-Oxo-6-(4-piperidinium-4-yl-butyrylamino)-1,3-dihyd-
roisoindol-2-yl]acetic acid trifluoroacetate (17b). Mp = 114–
115 °C; 1H NMR
d (400 MHz, d6-DMSO) 0.75–0.85 (m, 4H),
1.02–1.11 (m, 1H), 1.16 (dt, J = 14.9, 7.5 Hz, 2H), 1.35 (d,
J = 13.4 Hz, 2H), 1.88 (t, J = 7.2 Hz, 2H), 2.37 (dd, J = 32.5, 11.9 Hz,
1H), 2.80 (d, J = 12.2 Hz, 2H), 3.81 (s, 2H), 3.99 (s, 2H), 7.06 (d,
J = 8.3 Hz, 1H), 7.24 (dd, J = 8.2, 1.1 Hz, 1H), 7.64 (s, 1H), 7.86 (d,
J = 8.1 Hz, 1H), 8.17 (d, J = 8.1 Hz, 1H), 9.71 (s, 1H); HRMS (FAB) m/z
calcd for C19H26N3O4 [M+H]+: 360.4368, found: 360.4374.
C
24H34N3O6 [M+H]+: 460.5551, found: 459.5559.
4.1.8.19. 4-{6-[4-(1-Boc-piperidin-4-yl)butyrylamino]-1-oxo-1,3-
dihydroisoindol-2-yl}butyric acid (16s).
Mp = 190–191 °C;
1H NMR d (400 MHz, d6-DMSO) 0.94 (ddd, J = 24.4, 12.3, 3.9 Hz,
2H), 1.20–1.26 (m, 2H), 1.38 (s, 9H), 1.42–1.44 (m, 1H), 1.60–1.65
(m, 4H), 1.82 (dt, J = 14.2, 7.1 Hz, 2H), 2.24 (t, J = 7.2 Hz, 2H), 2.31
(t, J = 7.3 Hz, 2H), 2.63–2.72 (m, 2H), 3.51 (t, J = 7.0 Hz, 2H), 3.91
(d, J = 10.3 Hz, 2H), 4.40 (s, 2H), 7.49 (d, J = 8.3 Hz, 1H), 7.68 (dd,
J = 8.1, 1.5 Hz, 1H), 8.02 (s, 1H), 10.08 (s, 1H), 12.09 (br s, 1H);
4.1.9.3. [1-Oxo-6-(4-piperidinium-4-yl-benzoylamino)-1,3-dihy-
droisoindol-2-yl]acetic acid trifluoroacetate (17C).
Mp
>300 °C; 1H NMR d (400 MHz, d6-DMSO) 1.84 (ddd, J = 26.7, 12.5,
2.9 Hz, 2H), 1.99 (d, J = 13.2 Hz, 2H), 2.93–2.98 (m, 1H), 3.04 (dd,
J = 23.5, 12.2 Hz, 2H), 3.41 (d, J = 11.7 Hz, 2H), 4.30 (s, 2H), 4.50

4658
A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
(s, 2H), 7.41 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 8.3 Hz, 1H), 7.95–7.99 (m,
3H), 8.23 (d, J = 1.5 Hz, 1H), 8.42 (dd, J = 21.3, 10.8 Hz, 1H), 8.69 (d,
J = 11.7 Hz, 1H), 10.44 (s, 1H); 13C NMR d (125 MHz, d6-DMSO)
29.67 (2C), 39.25 (1C), 43.98 (1C), 44.05 (2C), 50.46 (1C), 114.75
4.1.9.9. 3-[1-Oxo-6-(4-piperazinium-1-yl-benzoylamino)-1,3-dihyd-
roisoindol-2-yl]propionic acid trifluoroacetate (17i). Mp = 212–
214 °C; 1H NMR d (400 MHz, d6-DMSO + CCl4) 2.62 (t, J = 6.6 Hz,
2H), 3.27 (s, 4H), 3.53 (s, 4H), 3.74 (t, J = 6.6 Hz, 2H), 4.46 (s, 2H),
7.10 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.3 Hz, 1H), 7.93–7.99 (m, 3H),
8.19 (s, 1H), 9.02 (s, 2H), 10.21 (s, 1H); 13C NMR d (125 MHz, d6-
DMSO) 33.39 (1C), 38.61 (1C), 42.98 (2C), 44.89 (2C), 50.01 (1C),
(1C), 116.89 (1C, CF3COÀ), 124.08 (1C), 124.22 (1C), 127.06 (2C),
2
128.58 (2C), 132.60 (1C), 133.56 (1C), 137.50 (1C), 139.58 (1C),
148.86 (1C), 158.77 (1C, CF3COÀ), 165.95 (1C), 168.18 (1C),
2
170.99 (1C); HRMS (FAB) m/z calcd for C22H24N3O4 [M+H]+:
114.50 (1C), 114.72 (2C), 116.68 (1C, CF3COÀ), 123.83 (1C), 123.86
2
394.4543, found: 394.4545.
(1C), 129.69 (2C), 133.15 (1C), 136.94 (1C), 139.76 (1C), 152.61
(1C), 158.57 (1C, CF3COÀ), 165.44 (1C), 167.74 (1C), 173.29 (1C);
2
4.1.9.4. [1-Oxo-6-(4-piperazinium-1-yl-benzoylamino)-1,3-dihyd-
roisoindol-2-yl]acetic acid trifluoroacetate (17d). Mp = 241–
242 °C; 1H NMR d (500 MHz, d6-DMSO) 3.27 (s, 4H) 3.54 (s, 4H)
4.30 (s, 2H) 4.49 (s, 2H) 7.11 (d, J = 8.3 Hz, 2H) 7.58 (d, J = 8.1 Hz,
1H) 7.96–7.99 (m, 3H) 8.23 (s, 1H) 8.96 (s, 2H) 10.23 (s, 1H); 13C
NMR d (125 MHz, d6-DMSO) 42.98 (2C), 43.97 (1C), 44.90 (2C),
HRMS (FAB) m/z calcd for C22H25N4O4 [M+H]+: 409.4689, found:
409.4696.
4.1.9.10. 3-{1-Oxo-6-[(1,2,3,4-tetrahydroisoquinolinium-7-car-
bonyl)amino]-1,3-dihydroisoindol-2-yl}-propionic acid chlo-
ride (17j).
Mp = 276–277.5 °C; 1H NMR d (400 MHz, d6-DMSO)
50.45 (1C), 114.70 (1C), 114.73 (2C), 116.67 (1C, CF3COÀ), 123.97
2.63 (t, J = 7.0 Hz, 2H), 3.10 (t, J = 5.9 Hz, 2H), 3.40 (dd, J = 6.5,
4.4 Hz, 2H), 3.74 (t, J = 7.0 Hz, 2H), 4.35 (s, 2H), 4.48 (s, 2H), 7.40 (d,
J = 8.6 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.89–7.92 (m, 2H), 7.97 (dd,
J = 8.3, 1.7 Hz, 1H), 8.21 (d, J = 1.2 Hz, 1H), 9.67 (s, 2H), 10.54 (s,
1H); HRMS (FAB) m/z calcd for C21H22N3O4 [M+H]+: 380.4272, found:
380.4281.
2
(1C), 124.19 (1C), 125.11 (1C), 129.70 (2C), 132.55 (1C), 137.15
(1C), 139.84 (1C), 152.63 (1C), 158.75 (1C, CF3COÀ), 165.47 (1C),
2
168.23 (1C), 171.01 (1C); HRMS (FAB) m/z calcd for C21H23N4O4
[M+H]+: 395.4419, found: 366.4422.
4.1.9.5.
bonyl)amino]-1,3-dihydroisoindol-2-yl}acetic acid chloride
(17e).
Mp = 297.5–298 °C (decomposes); 1H NMR d (400 MHz,
d6-DMSO) 3.11 (t, J = 6.0 Hz, 2H), 3.40 (dd, J = 10.5, 6.3 Hz, 2H),
4.30 (s, 2H), 4.35 (t, J = 4.0 Hz, 2H), 4.50 (s, 2H), 7.40 (d, J = 8.3 Hz,
1H), 7.60 (d, J = 8.3 Hz, 1H), 7.90–7.92 (m, 2H), 8.01 (dd, J = 8.3,
1.7 Hz, 1H), 8.26 (d, J = 1.7 Hz, 1H), 9.76 (s, 2H), 10.59 (s, 1H); HRMS
(FAB) m/z calcd for C20H20N3O4 [M+H]+: 366.4001, found: 366.3997.
{1-Oxo-6-[(1,2,3,4-tetrahydroisoquinolinium-7-car-
4.1.9.11. 3-[1-Oxo-6-(3-piperazinium-1-yl-benzoylamino)-1,3-di-
hydroisoindol-2-yl]propionic
acid
trifluoroacetate
(17k). Mp = 102–103 °C; 1H NMR d (500 MHz, d6-DMSO) 2.63
(t, J = = 7.1 Hz, 2H), 3.29 (s, 4H), 3.46 (t, J = 4.8 Hz, 4H), 3.75 (t,
J = 6.8 Hz, 2H), 4.48 (s, 2H), 7.24 (d, J = 8.3 Hz, 1H), 7.43 (t,
J = 7.7 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.52 (s, 1H), 7.58 (d,
J = 8.1 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 8.17 (s, 1H), 8.88 (s, 2H),
10.39 (s, 1H); 13C NMR d (125 MHz, d6-DMSO) 33.39 (1C), 38.62
(1C), 43.17 (2C), 45.93 (2C), 50.04 (1C), 114.69 (1C), 115.37 (1C),
4.1.9.6. 3-[1-Oxo-6-(2-piperidinium-4-yl-acetylamino)-1,3-dihyd-
roisoindol-2-yl]propionic acid trifluoroacetate (17f). Mp = 112–
114 °C; 1H NMR d (500 MHz, d6-DMSO) 1.40 (dd, J = 24.9, 13.2 Hz,
2H) 1.85 (d, J = 15.1 Hz, 2H) 2.03–2.14 (m, 1H), 2.33 (d, J = 7.0 Hz,
2H), 2.61 (t, J = 6.8 Hz, 2H), 2.91 (dd, J = 22.8, 11.5 Hz, 2H), 3.27 (d,
J = 11.9 Hz, 2H), 3.73 (t, J = 6.8 Hz, 2H), 4.44 (s, 2H), 7.51
(d, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 8.30 (d,
J = 10.1 Hz, 1H), 8.59 (d, J = 7.0 Hz, 1H), 10.19 (s, 1H); 13C NMR d
(125 MHz, d6-DMSO) 28.65 (2C), 31.16 (1C), 33.36 (1C), 38.60 (1C),
116.09 (1C, CF3COÀ), 119.66 (1C), 119.78 (1C), 123.93 (1C), 124.01
2
(1C), 129.72 (1C), 133.21 (1C), 136.18 (1C), 137.34 (1C), 139.44
(1C), 150.46 (1C), 158.76 (1C, CF3COÀ), 166.30 (1C), 167.67 (1C),
2
173.28 (1C); HRMS (FAB) m/z calcd for C22H25N4O4 [M+H]+:
409.4689, found: 409.4687.
4.1.9.12.
1,3-dihydroisoindol-2-yl]butyric
(17l).
Glassy substance; 1H NMR d (500 MHz, d6-DMSO) 1.27 (d,
3-[1-Oxo-6-(2-piperidinium-4-yl-acetylamino)-
acid trifluoroacetate
43.04 (1C), 43.59 (2C), 49.97 (1C), 113.42 (1C), 116.58 (1C, CF3COÀ),
2
122.76 (1C), 124.02 (1C), 133.25 (1C), 136.85 (1C), 139.34 (1C),
J = 6.7 Hz, 3H), 1.37–1.44 (m, 2H), 1.85 (d, J = 13.5 Hz, 2H), 2.05–
2.12 (m, 1H), 2.33 (d, J = 6.7 Hz, 2H), 2.62 (ddd, J = 30.8, 15.3,
7.4 Hz, 2H), 2.92 (dd, J = 22.8, 11.9 Hz, 2H), 3.27 (d, J = 11.9 Hz,
2H), 4.39 (dd, J = 27.8, 17.1 Hz, 2H), 4.59 (dt, J = 14.4, 7.5 Hz, 1H),
7.52 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 8.04 (s, 1H), 8.35
(d, J = 7.3 Hz, 1H), 8.62 (d, J = 7.0 Hz, 1H), 10.20 (s, 1H); HRMS
158.74 (1C, CF3COÀ), 167.64 (1C), 170.31 (1C), 173.26 (1C); HRMS
2
(FAB) m/z calcd for
C
18H24N3O4 [M+H]+: 346.4097, found:
374.4690.
4.1.9.7. 3-[1-Oxo-6-(4-piperidinium-4-yl-butyrylamino)-1,3-dihyd-
roisoindol-2-yl]propionic acid trifluoroacetate (17g). Mp = 196–
(FAB) m/z calcd for
C
19H26N3O4 [M+H]+: 360.4368, found:
197 °C; 1H NMR
d (400 MHz, d6-DMSO) 1.20–1.30 (m, 4H),
360.4660.
1.49–1.55 (m, 1H), 1.58–165 (m, 2H), 1.81 (d, J = 12.9 Hz, 2H),
2.34 (t, J = 6.6 Hz, 2H), 2.60 (t, J = 6.9 Hz, 2H), 2.83 (dd, J = 20.8,
10.0 Hz, 2H), 3.26 (d, J = 11.2 Hz, 2H), 3.72 (t, J = 6.9 Hz, 2H), 4.43
(s, 2H), 7.50 (d, J = 7.9 Hz, 1H), 7.67 (d, J = 7.5 Hz, 1H), 8.05 (s, 1H),
8.32 (s, 1H), 8.64 (s, 1H), 10.14 (s, 1H); HRMS (FAB) m/z calcd for
4.1.9.13. 3-[1-Oxo-6-(4-piperidinium-4-yl-butyrylamino)-1,3-
dihydroisoindol-2-yl]butyric acid chloride (17m). Mp = 231–
234 °C; 1H NMR d (500 MHz, d6-DMSO) 1.21–1.29 (m, 5H), 1.32
(dd, J = 23.1, 11.4 Hz, 2H), 1.49–1.56 (m, 1H), 1.61 (t, J = 6.6 Hz, 2H),
1.79 (d, J = 13.5 Hz, 2H), 2.36 (t, J = 5.8 Hz, 2H), 2.61 (ddd, J = 31.1,
15.1, 7.8 Hz, 2H), 2.80 (dd, J = 21.2, 10.5 Hz, 2H), 3.21 (d,
J = 11.2 Hz, 2H), 4.37 (dd, J = 26.7, 17.7 Hz, 2H), 4.59 (dd, J = 12.8,
6.4 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.74 (d, J = 7.0 Hz, 1H), 8.07 (s,
1H), 8.82 (d, J = 7.6 Hz, 1H), 9.09 (d, J = 7.6 Hz, 1H), 10.36 (s, 1H);
13C NMR d (125 MHz, d6-DMSO) 18.51 (1C), 22.02 (1C), 28.30 (2C),
32.86 (1C), 35.06 (1C), 36.33 (1C), 43.15 (2C), 44.43 (1C), 45.58
(1C), 112.87 (1C), 122.25 (1C), 123.48 (1C), 133.02 (1C), 136.05
(1C), 139.27 (1C), 166.82 (1C), 171.47 (1C), 172.25 (1C); HRMS
C
20H28N3O4 [M+H]+: 374.4639, found: 374.4643.
4.1.9.8. 3-[1-Oxo-6-(4-piperidinium-4-yl-benzoylamino)-1,3-dihyd-
roisoindol-2-yl]propionic acid trifluoroacetate (17h). Mp = 243–
244 °C (decomposes); 1H NMR d (400 MHz, d6-DMSO) 1.85 (dd,
J = 23.7, 11.5 Hz, 2H), 1.99 (d, J = 13.2 Hz, 2H), 2.63 (t, J = 7.0 Hz,
2H), 2.93–3.09 (m, 3H), 3.42 (d, J = 11.7 Hz, 2H), 3.75 (t, J = 7.0 Hz,
2H), 4.48 (s, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.57 (d, J = 8.3 Hz, 1H),
7.94 (dd, J = 8.3, 1.5 Hz, 1H), 7.97 (d, J = 8.1 Hz, 2H), 8.20 (d,
J = 1.5 Hz, 1H), 8.57 (s, 1H), 8.77 (t, J = 12.7, 2H), 10.43 (s, 1H);
HRMS (FAB) m/z calcd for C23H26N3O4 [M+H]+: 408.4814, found:
408.4816.
(FAB) m/z calcd for
C
21H30N3O4 [M+H]+: 388.4910, found:
388.4872.

A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
4659
4.1.9.14. 3-[1-Oxo-6-(4-piperidinium-4-yl-benzoylamino)-1,3-
dihydroisoindol-2-yl]butyric acid chloride (17n). Mp = 200–
4.1.9.19. 4-[1-Oxo-6-(4-piperidinium-4-yl-butyrylamino)-1,3-
dihydroisoindol-2-yl]butyric
(17s).
acid
trifluoroacetate
(400 MHz, d6-DMSO)
202 °C; 1H NMR d (500 MHz, d6-DMSO) 1.28 (d, J = 6.7 Hz, 3H),
1.94–2.02 (m, 4H), 2.64 (ddd, J = 31.4, 15.2, 7.4 Hz, 2H), 2.94–3.04
(m, 3H), 3.36 (d, J = 10.6 Hz, 2H), 4.43 (dd, J = 27.8, 17.4 Hz, 2H),
4.61 (td, J = 14.0, 7.0 Hz, 1H), 7.40 (d, J = 7.8 Hz, 2H), 7.56 (d,
J = 8.0 Hz, 1H), 7.97–8.01 (m, 3H), 8.20 (s, 1H), 9.23–9.27 (m, 1H),
9.32–9.34 (m, J = 9.6 Hz, 1H), 10.50 (s, 1H); HRMS (FAB) m/z calcd
for C24H28N3O4 [M+H]+: 422.5085, found: 422.5091.
Mp = 200–202 °C; 1H NMR
d
1.20–1.28 (m, 4H), 1.48–1.55 (m, 1H), 1.6 (dt, J = 15.0, 7.3 Hz,
2H), 1.78–1.86 (m, 4H), 2.24 (t, J = 7.2 Hz, 2H), 2.33 (t, J = 7.1 Hz,
2H), 2.83 (dd, J = 21.7, 10.9 Hz, 2H), 3.25 (d, J = 12.2 Hz, 2H), 3.51
(t, J = 6.8 Hz, 2H), 4.40 (s, 2H), 7.49 (d, J = 8.1 Hz, 1H), 7.66 (d,
J = 8.1 Hz, 1H), 8.04 (s, 1H), 8.27 (s, 1H), 8.68 (s, 1H), 10.13 (s,
1H); 13C NMR d (125 MHz, d6-DMSO) 22.41 (1C), 23.74 (1C),
28.87 (2C), 31.50 (1C), 33.30 (1C),35.49 (1C), 36.81 (1C), 41.69
(1C), 43.80 (2C), 49.59 (1C), 113.38 (1C), 116.44 (1C, CF3COÀ),
2
4.1.9.15. 3-[1-Oxo-6-(4-piperazinium-4-yl-benzoylamino)-1,3-
122.61 (1C), 123.96 (1C), 133.34 (1C), 136.61 (1C), 139.57 (1C),
157.75 (1C, CF3COÀ), 167.81 (1C), 171.78 (1C), 174.39 (1C); HRMS
dihydroisoindol-2-yl]butyric acid chloride (17o).
Mp = 258–
2
258.5 °C (decomposes); 1H NMR d (500 MHz, d6-DMSO) 1.28 (d,
J = 6.8 Hz, 3H), 2.64 (ddd, J = 31.2, 15.3, 7.1 Hz, 2H), 3.21 (s, 4H),
3.57 (t, J = 4.3, 4H), 4.41 (dd, J = 27.9, 17.6 Hz, 2H), 4.61 (td,
J = 13.6, 6.5 Hz, 1H), 7.10 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 8.3 Hz,
2H), 7.97 (m, J = 8.1 Hz, 3H), 8.19 (s, 1H), 9.51 (s, 2H), 10.26 (s,
1H); 13C NMR d (125 MHz, d6-DMSO) 18.99 (1C), 42.74 (2C),
44.76 (2C), 44.88 (1C), 46.06 (1C), 114.53 (2C), 114.69 (1C),
123.83 (1C), 123.85 (1C), 125.03 (1C), 129.73 (2C), 133.42
(1C), 136.84 (1C), 139.80 (1C), 152.63 (1C), 165.45 (1C), 167.25
(1C), 172.71 (1C); HRMS (FAB) m/z calcd for C23H27N4O4 [M+H]+:
423.4960, found: 423.4953.
(FAB) m/z calcd for C21H30N3O4 [M+H]+: 388.4910, found:
388.4904.
4.1.9.20. 4-[1-Oxo-6-(4-piperidinium-4-yl-benzoylamino)-1,3-
dihydroisoindol-2-yl]butyric
(17t).
acid
trifluoroacetate
Mp = 280–281 °C; 1H NMR d (400 MHz, d6-DMSO)
1.79–1.90 (m, 4H), 2.00 (d, J = 12.7 Hz, 2H), 2.26 (t, J = 6.6 Hz,
2H), 2.93–3.08 (m, 3H), 3.40–3.43 (m, 2H), 3.54 (t, J = 6.5 Hz, 2H),
4.45 (s, 2H), 7.40 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 7.6 Hz, 2H), 7.92–
7.97 (m, 3H), 8.18 (s, 1H), 8.52 (s, 1H), 8.77 (s, 1H), 10.43 (s, 1H);
13C NMR d (125 MHz, d6-DMSO) 23.76 (1C), 29.67 (2C), 31.53
(1C), 39.25 (1C), 41.72 (1C), 44.05 (2C), 49.66 (1C), 114.65 (1C),
116.72 (1C, CF3COÀ), 123.83 (1C), 123.93 (1C), 127.05 (2C),
4.1.9.16. 3-{1-Oxo-6-[(1,2,3,4-tetrahydroisoquinolinium-7-car-
bonyl)amino]-1,3-dihydroisoindol-2-yl}butyric acid chloride
2
128.57 (2C), 133.31 (1C), 133.58 (1C), 137.27 (1C), 139.48 (1C),
148.85 (1C), 158.77 (1C, CF3CCOÀ), 165.91 (1C), 167.81 (1C),
(17p).
Hygroscopic substance; 1H NMR
d (500 MHz, d6-
2
174.41 (1C); HRMS (FAB) m/z calcd for C24H28N3O4 [M+H]+:
DMSO) 1.29 (d, J = 6.6 Hz, 3H), 2.64 (ddd, J = 30.8, 15.2, 7.1 Hz,
2H), 3.09 (t, J = 6.0 Hz, 2H), 3.45 (d, J = 5.1 Hz, 2H), 4.39–4.48 (m,
4H), 4.62 (td, J = 14.2, 7.3 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.59 (d,
Jv8.6 Hz, 1H), 7.88–7.95 (m, 3H), 8.18 (s, 1H), 9.17 (s, 2H), 10.46
(s, 1H); HRMS (FAB) m/z calcd for C22H24N3O4 [M+H]+: 394.4543,
found: 394.4546.
422.5085, found: 422.5088.
4.1.9.21. 4-{1-Oxo-6-[(1,2,3,4-tetrahydroisoquinolinium-7-car-
bonyl)amino]-1,3-dihydroisoindol-2-yl}butyric acid chloride
(17u).
Hygroscopic substance; 1H NMR
d (400 MHz, d6-
DMSO) 1.86 (dt, J = 13.7, 6.7 Hz, 2H), 2.27 (t, J = 6.7 Hz, 2H), 3.09
(t, J = 5.2 Hz, 2H), 3.43–3.49 (m, 2H), 3.55 (t, J = 6.7 Hz, 2H), 4.40
(s, 2H), 4.46 (s, 2H), 7.42 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 8.3 Hz,
1H), 7.88 (s, 1H), 7.90–7.96 (m, 2H), 8.19 (s, 1H), 9.21 (s, 2H),
10.46 (s, 1H); HRMS (FAB) m/z calcd for C22H24N3O4 [M+H]+:
394.4543, found: 394.4540.
4.1.9.17. 3-[1-Oxo-6-(3-piperazinium-4-yl-benzoylamino)-1,3-
dihydroisoindol-2-yl]butyric acid chloride (17q).
Mp = 232–
233 °C; 1H NMR d (500 MHz, d6-DMSO) 1.28 (d, J = 5.4 Hz, 3H),
2.64 (ddd, J = 30.6, 14.8, 7.8 Hz, 2H), 3.24 (s, 4H), 3.52 (s, 4H),
4.42 (dd, J = 26.5, 17.4 Hz, 2H), 4.59–4.65 (m, 1H), 7.23 (d,
J = 7.5 Hz, 1H), 7.41 (t, J = 6.7 Hz, 1H), 7.50 (d, J = 6.5 Hz, 1H), 7.56
(d, J = 7.8 Hz, 1H), 7.60 (s, 1H), 7.99 (d, J = 7.3 Hz, 1H), 8.20 (s,
1H) 9.53 (s, 2H), 10.52 (s, 1H); 13C NMR d (125 MHz, d6-DMSO)
18.99 (1C), 38.72 (1C), 42.92 (2C), 44.90 (1C), 45.81 (2C), 46.10
(1C), 114.80 (1C), 115.49 (1C), 119.75 (1C), 123.91 (1C), 124.12
(1C), 129.69 (1C), 133.43 (1C), 136.08 (1C), 137.24 (1C), 139.49
(1C), 150.42 (1C), 166.28 (1C), 167.20 (1C), 172.70 (1C); HRMS
4.2. X-ray diffraction
The X-ray data for 5b, 14, 17c–TfaOHÁ5H2O, 17h-TfaOHÁ2H2O,
and 17tÁH2O were collected utilizing MoK
a radiation at Nonius
kappa CCD diffractometer at 100 K, and at Xcalibur Oxford Diffrac-
tion CCD diffractometer at room temperature for 17c, 17h, 17iÁH2O,
and 17jÁH2O. Final unit cell dimensions were obtained and refined
on an entire data set. All calculations to solve the structures and to
refine the models were carried out with the programs SHELXS97 and
(FAB) m/z calcd for
C
23H27N4O4 [M+H]+: 423.4960, found:
423.4967.
SHELXL97.
22 In all structures nonhydrogen nondisordered atoms have
4.1.9.18.
dihydroisoindol-2-yl]butyric
4-[1-Oxo-6-(2-piperidinium-4-yl-acetylamino)-1,3-
acid trifluoroacetate (17r).
been refined with anisotropic displacement parameters. In 17c the
Tfa anion is disordered over two positions with the occupancies
0.876(3) and 0.124(4). The F atoms in the minor component were
refined in isotropic approximation. In 17iÁH2O the fluorine atoms
in the Tfa anion are disordered over three positions with the occu-
pancies 0.756(12), 0.156(10) and 0.088(7). The minor components
were refined in isotropic approximation. The X-ray data for
17jÁH2O obtained from the poor-diffracting crystal revealed the
disordering of chloride anion and water molecule. For both of these
disordered species five close proximal positions with different
occupancies have been found and refined in isotropic approxima-
tion with the combined occupancies of unity, both for Cl anion
and H2O. In all structures the C-bound H atoms were placed in cal-
Mp = 184–185 °C; 1H NMR d (500 MHz, d6-DMSO) 1.37–1.45 (m,
2H), 1.80–1.87 (m, 4H), 2.05–2.12 (m, 1H), 2.25 (t, J = 6.9 Hz, 2H),
2.33 (d, J = 6.7 Hz, 2H), 2.91 (dd, J = 23.0, 11.2 Hz, 2H), 3.28 (d,
J = 11.2 Hz, 2H), 3.53 (t, J = 6.9 Hz, 2H), 4.41 (s, 2H), 7.51 (d,
J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 8.05 (s, 1H), 8.33–8.38 (m,
1H), 8.61–8.66 (m, 1H), 10.20 (s, 1H); 13C NMR d (125 MHz, d6-
DMSO) 23.72 (1C), 28.65 (2C), 31.17 (1C), 31.47 (1C), 41.66 (1C),
43.05 (1C), 43.58 (2C), 49.58 (1C), 113.49 (1C), 116.17 (1C,
CF3COÀ), 122.68 (1C), 124.02 (1C), 133.34 (1C), 136.83 (1C),
2
139.33 (1C), 158.56 (1C, CF3COÀ), 167.76 (1C), 170.31 (1C),
2
174.42 (1C); HRMS (FAB) m/z calcd for C19H26N3O4 [M+H]+:
360.4368, found: 360.4372.

4660
A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
culated positions and were treated in a riding model approxima-
tion with Uiso(H) = 1.2 Ueq(C), the O- and N-bound H-atoms were
found from difference Fourier maps and refined with isotropic dis-
placement parameters Uiso(H) = 1.5 Ueq(O), Uiso(H) = 1.2 Ueq(N). The
Figures were produced using Mercury.23 CCDC 912329–912337
contain the crystallographic data for studied compounds. These
data can be obtained free of charge from the Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
4.2.5. Crystal data and structure refinement parameters for 17h
Empirical formula: C25H26F3N3O6 formula weight: 521.49. Crys-
tal system: monoclinic. Space group: P21/c. Unit cell dimensions:
a = 13.675(4), b = 6.202(8), c = 30.779(17) Å, b = 115.42(3)°,
V = 2358(3) Å3.
Index
ranges:
À14 6 h 6 14,
À6 6 k 6 6,
À32 6 l 6 31. Range for data collection from 1.65° to 22.06°,
Z = 4. Dcalc = 1.469 mg/m3. F(000) = 1088. Reflections collected
4516. Independent reflections 2242 [R(int) = 0.1700]. Data/re-
straints/parameters 2242:228:316. Refinement method: Full ma-
trix least squares on F2. Goodness-of-fit on F2: 1.133. Final R
indices [I > 2(I)]: R1 = 0.2046, wR2 = 0.3531. R indices (all data):
R1 = 0.2967, wR2 = 0.3908. Largest diff. peak and hole: 0.516 and
4.2.1. Crystal data and structure refinement parameters for 5b
Empirical formula: C12H14N2O3 formula weight: 234.25. Crystal
system: monoclinic. Space group: P21/n. Unit cell dimensions:
a = 5.9940(3), b = 8.1340(5), c = 22.2920(13), b = 96.223(2)°,
V = 1080.45(11) Å3. Index ranges: À7 6 h 6 7, À10 6 k 6 9,
À23 6 l 6 28. Range for data collection from 1.84° to 26.99°, Z = 4.
Dcalc = 1.440 mg/m3. F(000) = 496. Reflections collected 6247. Inde-
pendent reflections 2360 [R(int) = 0.0429]. Data/restraints/parame-
ters2360:0:163. Refinementmethod:FullmatrixleastsquaresonF2.
Goodness-of-fit on F2: 1.006. Final R indices [I > 2(I)]: R1 = 0.0493,
wR2 = 0.1350. R indices (all data): R1 = 0.0666, wR2 = 0.1464. Largest
À0.414 e ÅÀ3
.
4.2.6. Crystal data and structure refinement parameters for
3-[1-oxo-6-(4-piperidinium-4-yl-benzoylamino)-1,3-
dihydroisoindol-2-yl]propionate dihydrate (17h-TfaOHÁ2H2O)
Empirical formula: C23H29N3O6 formula weight: 443.49. Crystal
system: triclinic. Space group: P-1. Unit cell dimensions:
a = 7.8610(4), b = 10.0294(4), c = 15.0560(5) Å,
a = 96.388(2),
diff. peak and hole: 0.285 and À0.286 e ÅÀ3
.
b = 99.306(2),
c
= 111.4031(18)°, V = 1071.73(8) Å3. Index ranges:
À9 6 h 6 9, À12 6 k 6 12, À18 6 l 6 18. Range for data collection
from 2.22° to 25.49°, Z = 2. Dcalc = 1.374 mg/m3. F(000) = 472.
Reflections collected 7408. Independent reflections 3961
[R(int) = 0.0525]. Data/restraints/parameters 3961:0:310. Refine-
ment method: Full matrix least squares on F2. Goodness-of-fit on
F2: 1.007. Final R indices [I > 2(I)]: R1 = 0.0696, wR2 = 0.1778. R indi-
ces (all data): R1 = 0.0923, wR2 = 0.1913. Largest diff. peak and
4.2.2. Crystal data and structure refinement parameters for 14
Empirical formula: C15H19NO4 formula weight: 277.31. Crystal
system: monoclinic. Space group: P21/c. Unit cell dimensions:
a = 11.9373(9), b = 10.3546(8), c = 11.5307(9), b = 102.543(4)°,
V = 1391.25(19) Å3. Index ranges: À15 6 h 6 15, À12 6 k 6 13,
À14 6 l 6 14. Range for data collection from 2.63° to 27.00°, Z = 4.
Dcalc = 1.324 mg/m3. F(000) = 592. Reflections collected 5573. Inde-
pendent reflections 3015 [R(int) = 0.0541]. Data/restraints/parame-
ters3015:0:188. Refinementmethod:FullmatrixleastsquaresonF2.
Goodness-of-fit on F2: 1.006. Final R indices [I > 2(I)]: R1 = 0.0633,
wR2 = 0.1400. R indices (all data): R1 = 0.0910, wR2 = 0.1546. Largest
hole: 0.400 and À0.284 e ÅÀ3
.
4.2.7. Crystal data and structure refinement parameters for
3-[1-oxo-6-(4-piperazinium-1-yl-benzoylamino)-1,3-
dihydroisoindol-2-yl]propionic acid trifluoroacetate hydrate
(17iÁH2O)
diff. peak and hole: 0.248 and À0.309 e ÅÀ3
.
Empirical formula: C24H27F3N4O7 formula weight: 540.50. Crys-
tal system: triclinic. Space group: P-1. Unit cell dimensions:
4.2.3. Crystal data and structure refinement parameters for
[1-oxo-6-(4-piperidinium-4-yl-benzoylamino)-1,3-dihydrois-
oindol-2-yl]acetate pentahydrate (17c-TfaOHÁ5H2O)
a = 8.9199(5), b = 11.8606(9), c = 12.0078(7) Å,
a = 92.329(5),
b = 93.603(5),
c
= 106.407(6)°, V = 1213.97(13) Å3. Index ranges:
À6 6 h 6 10, À14 6 k 6 13, À14 6 l 6 14. Range for data collection
from 3.02° to 25.05°, Z = 2. Dcalc = 1.479 mg/m3. F(000) = 564.
Reflections collected 7655. Independent reflections 4295
Empirical formula: C22H33N3O9 formula weight: 483.51. Crystal
system: monoclinic. Space group: P21/n. Unit cell dimensions:
a = 12.8160(3), b = 11.1350(3), c = 17.1290(5) Å, b = 108.7240(8)°,
V = 2315.04(11) Å3. Index ranges: À16 6 h 6 16, À13 6 k 6 13,
À21 6 l 6 21. Range for data collection from 2.40° to 26.50°, Z = 4.
Dcalc = 1.387 mg/m3. F(000) = 1032. Reflections collected 18213.
Independent reflections 4793 [R(int) = 0.0500]. Data/restraints/
parameters 4793:0:346. Refinement method: Full matrix least
squares on F2. Goodness-of-fit on F2: 1.003. Final R indices [I > 2(I)]:
[R(int) = 0.0251].
Refinement method: Full matrix least squares on F2. Goodness-
of-fit on F2: 1.004. Final
indices [I > 2(I)]: R1 = 0.0507,
Data/restraints/parameters
4295:109:383.
R
wR2 = 0.1147. R indices (all data): R1 = 0.0923, wR2 = 0.1241. Larg-
est diff. peak and hole: 0.495 and À0.278 e ÅÀ3
.
R1 = 0.0474, wR2 = 0.1117.
R indices (all data): R1 = 0.0596,
4.2.8. Crystal data and structure refinement parameters for 3-
{1-oxo-6-[(1,2,3,4-tetrahydroisoquinolinium-7-
wR2 = 0.1185. Largest diff. peak and hole: 0.364 and À0.366 e ÅÀ3
.
carbonyl)amino]-1,3-dihydroisoindol-2-yl}propionic acid
chloride hydrate (17jÁH2O)
4.2.4. Crystal data and structure refinement parameters for 17c
Empirical formula: C24H24F3N3O6 formula weight: 507.46. Crys-
tal system: triclinic. Space group: P-1. Unit cell dimensions:
Empirical formula: C21H22ClN3O5 formula weight: 431.87. Crys-
tal system: monoclinic. Space group: C2/c. Unit cell dimensions:
a = 46.068(7), b = 5.0936(7), c = 18.848(3) Å, b = 109.607(13)°,
V = 4166.3(11) Å3. Index ranges: À54 6 h 6 54, À6 6 k 6 5,
À22 6 l 6 18. Range for data collection from 2.98° to 25.05°,
Z = 8. Dcalc = 1.377 mg/m3. F(000) = 1808. Reflections collected
6442. Independent reflections 3648 [R(int) = 0.0647]. Data/re-
straints/parameters 3648:2:290. Refinement method: Full matrix
least squares on F2. Goodness-of-fit on F2: 1.004. Final R indices
a = 7.2240(8),
b = 93.852(4),
b = 9.8920(9),
c = 17.329(2),
a = 97.142(7),
c
= 97.901(7), V = 1212.6(2) Å3. Index ranges:
À8 6 h 6 8, À11 6 k 6 8, À20 6 l 6 20. Range for data collection
from 2.10° to 25.00°, Z = 2. Dcalc = 1.390 mg/m3. F(000) = 528.
Reflections collected 6908. Independent reflections 4135
[R(int) = 0.0374]. Data/restraints/parameters 4135:16:360. Refine-
ment method: Full matrix least squares on F2. Goodness-of-fit on
F2: 1.001. Final R indices [I > 2(I)]: R1 = 0.0781, wR2 = 0.1818. R indi-
ces (all data): R1 = 0.0972, wR2 = 0.1918. Largest diff. peak and
[I > 2(I)]: R1 = 0.0500, wR2 = 0.0917.
R
indices (all data):
R1 = 0.2115, wR2 = 0.1289. Largest diff. peak and hole: 0.209 and
À0.279 e ÅÀ3
.
hole: 0.639 and À0.459 e ÅÀ3
.

A. A. Krysko et al. / Bioorg. Med. Chem. 21 (2013) 4646–4661
4661
4.2.9. Crystal data and structure refinement parameters for
References and notes
4-[1-oxo-6-(4-piperidinium-4-yl-benzoylamino)-1,3-
dihydroisoindol-2-yl]butyric acid trifluoroacetate hydrate
(17tÁH2O)
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Empirical formula: C26H30F3N3O7 formula weight: 553.53. Crys-
tal system: triclinic. Space group: P-1. Unit cell dimensions:
a = 7.1440(6), b = 9.3970(6), c = 20.6680(16) Å,
a = 84.549(5),
b = 88.466(5),
c
= 69.296(5)°, V = 1291.98(17) Å3. Index ranges:
À7 6 h 6 7, À10 6 k 6 10, À22 6 l 6 22. Range for data collection
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1.011. Final R indices [I > 2(I)]: R1 = 0.0887, wR2 = 0.1985. R indices
(all data): R1 = 0.1432, wR2 = 0.2232. Largest diff. peak and hole:
0.345 and À0.342 e ÅÀ3
.
Acknowledgments
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This article is dedicated to the memory of Dr. Yurii A. Simonov,
who took active part in this research.
The authors are grateful to Dr. V.V. Polovinko (Enamine LTD,
Ukraine) for the 1H NMR spectra. We also thank Dr. A.V. Mazepa
and Dr. I.M. Rakipov (A.V. Bogatsky Physico-Chemical Institute of
the National Academy of Sciences of Ukraine) for the Mass spectra.
T.M. Khristova thanks the French Embassy in Ukraine for a PhD
fellowship.
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Supplementary data
22. Sheldrick, G. M. Acta Cryst. 2008, A64, 112.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.019.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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