Cyclosquaramides as Kinase Inhibitors with Anticancer Activity

Article abstract of DOI:10.1002/cmdc.201200157

We report the synthesis and biological evaluation of a new series of oligosquaramide-based macrocycles as anticancer agents. Compound 7, considered as representative of this series, exhibited significant antiproliferative activity against the NCI-60 human tumor cell line panel, with IC₅₀ values ranging from 1 to 10μM. The results show that sensitivity to cyclosquaramides is clearly dependent on cell type, underscoring a degree of biological selectivity. The observed antiproliferative effects appear to be related to deregulation of protein phosphorylation, as compounds 7 and 8 are effective inhibitors of several important kinases such as ABL1, CDK4, CHK1, PKC, c-MET, and FGFR, among others. The corresponding acyclic oligosquaramides and smaller cyclosquaramides did not show antitumor activity, suggesting that a macrocyclic structure with minimal molecular size plays a key role in the observed antitumor activity.

Full text of DOI:10.1002/cmdc.201200157

MED
DOI: 10.1002/cmdc.201200157
Cyclosquaramides as Kinase Inhibitors with Anticancer
Activity
Priam Villalonga,[b] Silvia Fernꢀndez de Mattos,[b] Guillem Ramis,[b] Antꢁnia Obrador-Hevia,[b]
Angel Sampedro,[a] Carmen Rotger,*[a] and Antoni Costa*[a]
We report the synthesis and biological evaluation of a new
series of oligosquaramide-based macrocycles as anticancer
agents. Compound 7, considered as representative of this
series, exhibited significant antiproliferative activity against the
NCI-60 human tumor cell line panel, with IC50 values ranging
from 1 to 10 mm. The results show that sensitivity to cyclo-
squaramides is clearly dependent on cell type, underscoring
a degree of biological selectivity. The observed antiproliferative
effects appear to be related to deregulation of protein phos-
phorylation, as compounds 7 and 8 are effective inhibitors of
several important kinases such as ABL1, CDK4, CHK1, PKC, c-
MET, and FGFR, among others. The corresponding acyclic oli-
gosquaramides and smaller cyclosquaramides did not show an-
titumor activity, suggesting that a macrocyclic structure with
minimal molecular size plays a key role in the observed antitu-
mor activity.
Introduction
In recent years, squaramides have emerged as promising phar-
macophores for drug design. The biological activity of squara-
mides relies on specific structural features; the squaramide[1]
motif provides a planar aromatic[2] framework equipped with
two adjacent carbonyl acceptor groups and two NH amide-
type donor sites, capable of establishing multiple hydrogen
bond interactions.[3–6] A number of squaramides are currently
under investigation for biological effects such as bioisosteric
substitution of phosphate groups in modified oligodeoxynu-
cleotides[7,8] and of guanidines in compounds that show activi-
ty as potassium channel openers.[9–11] In addition, squaramide-
based compounds target certain CXC-type chemokine recep-
tors[12–14] and are involved in ligands designed for receptor-
binding inhibition of enterotoxins.[15–17] Moreover, recent re-
ports have also demonstrated the anticancer activity of certain
squaramide compounds.[18,19]
with a certain degree of conformational pre-organization and
flexibility very well suited for ligand binding on target surfaces
with potential antitumor effects.[23] This compromise between
pre-organization and flexibility is a key characteristic of macro-
cyclic cyclopeptides with known anticancer activity.[24–27] We
anticipated that being nonpeptidic agents, cyclosquaramides
could resist degradation. Herein we present a series of novel
cyclosquaramides, and we report data that confirm the anti-
cancer and kinase inhibitory activities of these compounds
(Figure 1).
Results and Discussion
Synthesis
Despite several structural similarities with secondary amides,
squaramides have their own unique structural features. In solu-
tion, secondary squaramides show a similar, although less pro-
nounced, preference for the anti isomer as do secondary
amides. The free energy difference between the two rotamers
is low (~0.25 kcalmolÀ1). As in amides, the CÀN bond of secon-
dary squaramides has double bond character, and the rotation-
al energy barrier (~12 kcalmolÀ1) is also relatively low com-
pared with that of secondary amides.[20] In fact, at sub-ambient
temperatures both the anti–anti and anti–syn (syn–anti) iso-
The synthesis of oligomeric cyclosquaramides 1–10 follows
a modular approach. Macrocycles 4–9 were prepared as de-
scribed previously by us,[22] obtaining linear squaramide com-
pounds of various length (Scheme 1) and condensing them in
a macrocyclization reaction either with one equivalent of di-
ethyl squarate or one equivalent of the diethyl ester 12 to
afford cyclosquaramides 4–9, respectively (Scheme 2).
[a] A. Sampedro, Dr. C. Rotger, Dr. A. Costa
Departament de Quꢀmica, Universitat de les Illes Balears
Carretera Valldemossa km 7.5 (Spain)
E-mail: carmen.rotger@uib.es
1
mers are detected by H NMR spectroscopy.[21] We also demon-
strated that the inclusion of tertiary amino groups as hydrogen
bond donors in the vicinity of the squaramido NH groups in-
duces formation of b-turn analogues in secondary squara-
mides, allowing the synthesis of oligomeric cyclosquaramides
through hydrogen-bond-directed macrocyclization.[22]
antoni.costa@uib.es
[b] Dr. P. Villalonga, Dr. S. Fernꢁndez de Mattos, G. Ramis, Dr. A. Obrador-Hevia
Institut Universitari d’Investigaciꢂ en Ciꢃncies de la Salut (IUNICS)
Universitat de les Illes Balears
Carretera Valldemossa km 7.5 (Spain)
Based on these precedents, we hypothesized that oligomeric
cyclosquaramides might provide well-defined architectures
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200157.
ChemMedChem 0000, 00, 1 – 10
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
1
&
ÞÞ
These are not the final page numbers!

C. Rotger, A. Costa, et al.
MED
macrocyclization to cyclosquaramides 1 and 2, respectively
(Scheme 2).
Similarly, cyclosquaramide 10 was prepared as described for
macrocycle 2. The diamine N1-(3-aminopropyl)-N1-(phenylmeth-
yl)-1,3-propanediamine was condensed with two equivalents
of 11 to afford the di-oligosquaramide compound 18. The de-
protection and condensation cycle was repeated to afford the
corresponding tetra-oligosquaramide compound 19. Deprotec-
tion of 19 followed by condensation with diethyl squarate af-
forded cyclosquaramide 9. Alternatively, cyclosquaramide 3
was prepared after deprotection of the tri-oligosquaramide 20
followed by its condensation with diethyl ester 17. See the
Supporting Information for an outline of the synthesis of cyclo-
squaramides 3 and 9. For all cases studied thus far, the macro-
cycles were obtained in remarkably good yields without using
high-dilution conditions.
Figure 1. Structures of oligomeric cyclosquaramides.
Biological evaluation
The anticancer activity of cyclosquaramides 4–9 was initially
evaluated using the mantle cell lymphoma (MCL) cell lines
Jeko-1 and Z-138. MCL is an aggressive subtype of non-Hodg-
kin lymphoma that is mostly incurable under current therapies;
MCL patients have a median survival of about three years. It is
characterized by the t(11;14)(q13;q32) chromosomal transloca-
tion, combined with deregulation of cell proliferation and sur-
vival pathways, chromosome instability, and disruption of the
DNA damage response pathways.[28] To determine whether the
synthesized oligomeric cyclosquaramides 4–9 could affect
cancer cell proliferation or survival, MCL cell lines Jeko-1 and
Z-138 were treated for 48 h and assayed for viability using a lu-
minescence-based ATP assay (Cell Titer-Glo, Promega).
Data from dose–response
Macrocycles 1–3 and 10 were synthesized according to this
procedure with minor modifications using the corresponding
diamines as starting material. As exemplified in Scheme 1, N-
Boc-protected l-methionine 13 was condensed with N-Boc-1,3-
diaminopropane via the agency of DCC–DMAP as coupling re-
agent. After deprotection of both Boc groups of 14 with aque-
ous acid, the resulting diamine was rapidly condensed with
squaramide 11, the growing module, to afford 15. Repetition
of a cycle of deprotection–condensation with 15 afforded the
linear squaramide derivative 16. Deprotection of 16 was fol-
lowed by condensation with diethyl squarate or diethyl ester
12, and subsequent intramolecular hydrogen-bond-directed
studies listed in Table 1 reveal
that cyclosquaramides display
antiproliferative activity against
both cell lines. However, there is
clear evidence for the depend-
ence of the observed antiproli-
ferative activity on the size of
the macrocycle. Whereas the
smaller macrocycles 4–6, respec-
tively featuring two, three, and
four squaramides, do not show
significant activity against Jeko-
1 cells (IC50 >200 mm), the larger
members of this series, namely
cyclosquaramides
7
(IC50 =
7.1 mm), 8 (IC50 =10 mm), and 9
(IC50 =17.9 mm), display activities
in the micromolar range.
This trend is also observed
with the Z-138 cell line. Plots of
the antiproliferative activity as
a function of the number of
squaramide units present in the
macrocycles after treatment at
Scheme 1. Synthetic procedure for the preparation of oligomeric squaramides.
&2
&
www.chemmedchem.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
ÝÝ
These are not the final page numbers!

Cyclosquaramides as Kinase Inhibitors
MED
selectivity. Importantly, cyclo-
squaramide 7 is consistently the
most active compound, even in
the least sensitive cell lines.
To further investigate how
structural modifications on the
cyclosquaramide scaffold affect
activity, we prepared some struc-
turally different cyclosquara-
mides containing five and six
squaramide units. The activities
of compounds 1–3 and 10 were
studied against Jeko-1 cells,
which proved to be more struc-
ture-dependent than Z-138 cells
regarding
cyclosquaramide-in-
duced antiproliferative activity
(Figure 2). As shown in Table 1,
modification of the aminoalkyl
linker into alkyl, benzyl, or me-
thionine fragments led to a mod-
erate loss in biological activity;
the activity of compound 7 is
higher than that observed for
modified cyclosquaramides 1–3
and 10.
Scheme 2. Synthetic procedure for the macrocyclization reaction.
Table 1. In vitro activity profiles of cyclosquaramides 1–9 against Jeko-
1 and Z-138 (MCL) cells.
Compd
Sq[a]
Mr [Da]
IC50 [mm][b]
Jeko-1
>200
>200
>200
Z-138
4
5
6
7
8
9
1
2
3
2
3
4
5
6
7
5
6
5
5
446.5
669.8
893.1
1116.3
1339.6
1584.9
1176.4
1199.7
1101.3
1192.4
27.3Æ5
21.2Æ7
5.1Æ0.9
4.7Æ0.9
6.6Æ1
7.0Æ1.3
–
7.1Æ1
10Æ2
18Æ2
34Æ6
24Æ4
45Æ5
27Æ5
–
–
–
10
[a] Number of squaramide units. [c] Data represent the meanÆSD (n=3);
all experiments were performed in triplicate.
*
Figure 2. Antiproliferative activity toward Jeko-1 (ꢀ) and Z-138 ( ) cells as
a function of the number of squaramide units upon treatment with cyclo-
squaramides 4–9 (10 mm, 48 h).
a compound concentration of 10 mm for 48 h shows that cyclo-
squaramide 7, formed by five squaramide units, is the most
active molecule of the series against both cell lines (Figure 2).
To further investigate the influence of size on cyclosquara-
mide activity, additional MCL cell lines (Granta-519 and Rec-1),
and cell lines of various tumor origin, such as glioblastoma
(U87MG and LN229) and osteosarcoma (U2OS) cells, were
treated with cyclosquaramides 7–9 at 10 mm for 48 h. As
shown in Figure 3, sensitivity to cyclosquaramides is clearly de-
pendent on cell type, which indicates a degree of biological
Among the physicochemical parameters that contribute to
tumor cell growth inhibitory activity, molecular weight has
proven to be one of the most important, together with rotata-
ble bonds, low polar surface area, and total hydrogen bond
count. From our data, it became clear that active cyclosquara-
mides are beyond the classical limitations used for assessing
oral bioavailability of drug candidates. The molecular weights
of the active compounds 1–3 and 7–10 are much higher than
ChemMedChem 0000, 00, 1 – 10
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
&
3
&
ÞÞ
These are not the final page numbers!

C. Rotger, A. Costa, et al.
MED
Figure 3. Percent cell viability upon treatment with cyclosquaramides 7–9
(10 mm) for 48 h relative to untreated cells: mantle cell lymphoma (Granta-
519 and Rec-1), glioblastoma (U87MG and LN229), and osteosarcoma
(U2OS); data represent the meanÆSD (n=3 independent experiments).
Figure 4. Percent cell viability upon treatment with cyclosquaramide 7
(10 mm, 48 h) relative to untreated cells. Cell lines of different tumor origins
used: colorectal cancer (SW620), osteosarcoma (U2OS), mantle cell lympho-
ma (Z-138, Jeko-1, Rec-1, and Granta-519), glioblastoma (U87MG, LN229,
T98G, and U373); data represent the mean ÆSD (n=3 independent experi-
ments).
the Mr <500 Da limit imposed by Lipinski rules. Higher Mr is
usually considered detrimental to in vivo potency. However,
these rules do not apply to many anticancer agents, of which
a majority have Mr >500 Da.[29] Therefore, new drugs can be
found within the high-Mr range, provided that the number of
rotatable bonds is minimized.[30]
Table 2. Cyclosquaramide 7 cytotoxicity determination against selected
tumor cell lines.[a]
Given our preliminary SAR data and considering that cyclo-
squaramide 7 is easily obtained at the multi-gram scale, we
used it as our reference compound to further investigate the
biological activity of cyclosquaramide compounds. The prelimi-
nary results obtained with cyclosquaramide 7 suggest the
presence of some cell-type selectivity. This assumption was
again confirmed by analyzing additional cell lines of various
tumor origin (Figure 4).
Cell line
Cell type
IC50 [mm][b]
5.07Æ0.75
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
IGR-OVI
NCI-H460
NCI-H522
HOP-92
NCI-H226
EKVX
ovarian carcinoma
ovarian carcinoma
ovarian carcinoma
ovarian carcinoma
ovarian carcinoma
ovarian carcinoma
lung carcinoma
lung carcinoma
lung carcinoma
lung carcinoma
lung carcinoma
colon carcinoma
colon carcinoma
colon carcinoma
colon carcinoma
colon carcinoma
melanoma
>10
>10
>10
3.10Æ0.12
2.38Æ0.15
6.88Æ1.25
The antitumor effect of cyclosquaramide 7 was also evaluat-
ed across the NCI-60 tumor cell line panel.[31] Compared with
the conventional anticancer agent paclitaxel, compound 7
showed mid-range activity at the low-micromolar range of IC50
in the NCI-60 panel. This screen allowed us to detect certain
selectivities. Thus, whereas a group of ovarian, lung, colon,
melanoma, and renal cancer cell lines did not respond to com-
pound 7 at the highest concentration tested (10 mm), other cell
lines of the same tumor origin were sensitive to the action of
7 (Table 2). Moreover, compound 7 did not exhibit toxicity
against control non-transformed cells, such as NIH3T3 fibro-
blasts (data not shown).
>10
3.53Æ0.24
1.53Æ0.60
2.75Æ0.15
2.98Æ0.15
HCT-116
HCT-15
HT-29
>10
>10
1.80Æ0.27
3.23Æ0.18
>10
>10
SW-480
COLO-205
SK-MEL-28
LOX-IMVI
SK-MEL-2
SK-MEL-5
UACC-62
786-O
melanoma
melanoma
melanoma
melanoma
renal carcinoma
renal carcinoma
3.29Æ0.09
2.75Æ0.03
1.34Æ0.18
>10
Because U87MG glioma cells, which are sensitive to com-
pound 7 (Figures 3 and 4), spontaneously form multicellular
tumor spheroids in culture,[32] we also investigated whether
compound 7 could prevent spheroid formation. Whereas con-
trol U87MG cells formed high numbers of large and dense
multicellular tumor spheroids, cells treated with compound 7
were largely resistant to spheroid formation (Figure 5). These
observations confirm that this compound severely decreases
glioma cell proliferation.
TK-10
3.36Æ1.00
[a] See the Supporting Information for a complete list of IC50 values.
[b] Data represent the meanÆSD of three independent experiments per-
formed in triplicate.
tion by flow cytometry (Figure 6). Treatment of the MCL cell
lines Jeko-1 and JVM-2 with compound 7 (10 mm) for 24 h
caused distinct effects on both cell lines. In Jeko-1 cells, 7 in-
duced an increase in the sub-G1 fraction from 12.1 to 27.3%,
in parallel with a decrease in the S-phase fraction. Under the
To determine if cyclosquaramide treatment induces changes
in the cell-cycle profile, we examined cell-cycle phase distribu-
&4
&
www.chemmedchem.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
ÝÝ
These are not the final page numbers!

Cyclosquaramides as Kinase Inhibitors
MED
same conditions, JVM-2 cells were drastically affected by 7,
with an increment of cells in the sub-G1 population from 9.2 to
67.0%, indicating a dramatic induction of apoptosis.
Taken together, our results and the observed selectivities
toward the NCI-60 panel are compatible with a semi-selective
interaction of 7 with key biological targets. From this point of
view, targeting protein kinases is of interest, as deregulated
kinases are implicated in a variety of human diseases, and con-
sequently, a number of protein kinase inhibitors are in clinical
or preclinical evaluations as anticancer agents.[33,34] It is also
known that squaramide-based anticancer agents can involve
the inhibition of kinases. Examples include arylamidoaryl squar-
amides that inhibit KIT Tyr kinases,[18] and several heterocyclic
squaramides that inhibit checkpoint kinases Chk1 and Chk2:
two Ser/Thr regulatory kinases implicated in cell-cycle check-
point control.[19]
To determine whether cyclosquaramides can inhibit protein
kinases, the reference cyclosquaramide 7, which is active
against Jeko-1, JVM-2, and several cell lines within the NCI-60
panel, was initially tested at 10 mm in a single-point binding
assay against a panel of kinases (ProQinase, Freiburg, Germa-
ny).[35] The inhibitory activity, expressed as a percentage of con-
trol values (PoC<35), indicated that compound 7 inhibits 44
kinases over a panel of 210 representative kinases of the TK,
TKL, STE, CK1, AGC, CAMK, and CMGC families of the human
kinome (see Supporting Information). After this initial profile,
we decided to determine if the enzyme inhibitory activity is re-
lated to the in vitro antiproliferative activity. To this end, we
defined a subset of 25 kinases
Figure 5. Representative phase-contrast micrographs of U87MG cells left for
six days to allow formation of multicellular tumor spheroids (MCTS): untreat-
ed (control) or treated with 10 mm compound 7. The bar chart indicates the
values (meanÆSD) of MCTS formation from three independent experiments,
each conducted in duplicate, expressed as the percentage relative to un-
treated cells. The differences between control and cyclosquaramide treat-
ment are statistically significant (Student’s t-test: **p<0.01).
heavily affected by 7 in the ini-
tial profile. In these experiments,
the inhibitory potency was de-
termined against inactive cyclo-
squaramide 4 and active cyclo-
squaramides 7 and 8 at a single
concentration of 10 mm (Table 3).
These results strongly suggest
a relationship between in vitro
cellular antiproliferative activity
and kinase inhibitory potency.
Hence, compound 4, which is in-
active against Jeko-1 cells,
showed high PoC values, indicat-
ing low inhibitory potency,
whereas compounds 7 and 8
showed low PoC values. For ex-
ample, 7 leads to PoC<10 for
CSK, MATK, NLK, PKC-g, TRK-C
and ZAP-70.
In a separate experiment, the
inhibitory concentration (IC50)
was also calculated for reference
compound 7 (Table 3). In gener-
al, the enzymatic IC50 value lies
within
the
low-micromolar
Figure 6. Effect of cyclosquaramide 7 treatment on cell-cycle distribution for a) Jeko-1 and b) JVM-2 cells, assessed
by flow cytometry analysis upon propidium iodide staining. Representative histograms plotting cell events versus
DNA content (FL3 LIN) after treatment with 7 at 10 mm for 24 h.
range, thus confirming the inhib-
itory activity of cyclosquara-
ChemMedChem 0000, 00, 1 – 10
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
&
5
&
ÞÞ
These are not the final page numbers!

C. Rotger, A. Costa, et al.
MED
Conclusions
Table 3. Protein kinase affinity data for cyclosquaramides 4, 6, 7, and 8.
Kinase
Affinity [%][a]
IC50 [mm][b]
We have discovered cyclosquaramides as a novel class of
kinase inhibitors with anticancer activity. The compounds
tested so far showed significant (low-micromolar) inhibition of
kinases implied in cell proliferation such as ABL1, CDK4, PKC, c-
Met, and FGF-R, among others. Cyclosquaramide 7, used as ref-
erence compound, also demonstrated antiproliferative activity,
with IC50 values in the low-micromolar range in the NCI-60
screen and in two MCL cell lines. The observed IC50 values on
the same order of magnitude in both the enzyme- and cell-
based screens indicate that cyclosquaramides are cell-permea-
ble and that degradation does not take place to a considerable
extent. Although the biological target responsible for anticanc-
er activity remains unknown at this point, the information ob-
tained so far is compatible with a semi-selective interaction of
cyclosquaramides with conserved parts of target proteins, pre-
sumably kinases. Further research to elucidate the mechanism
of action and to determine the anticancer potential of cyclo-
squaramides is under way.
4
6
8
7
7
ABL1
ACK1
94
402
100
81
117
57
50
66
83
80
186
91
96
78
70
70
93
85
101
86
46
185
90
65
97
62
20
42
53
71
157
66
44
67
68
59
58
55
17
128
58
38
112
17
27
37
29
76
117
21
15
32
27
32
17
44
11
14
14
90
18
30
12
24
3
12
12
0
13
14
6
22
12
13
14
4
36
13
10
8
5.4Æ0.75
[c]
–
CDK4/CycD3
CDK5/p25NCK
CDK7/CycH/Mat1
CDK9/CycT
CHK1
5.3Æ0.50
9.2Æ0.70
28.0[d]
3.7Æ0.71
1.4Æ0.15
7.2Æ0.74
2.6Æ0.58
5.4Æ0.50
CSK
FGF-R1 VM
FGF-R2
MATK
c-MET
MUSK
NLK
PAK4
PAK7
PKC-a
PKC-b1
PKC-g
PLK1
S6K
TGFB-R1
TRK-C
[c]
–
3.7Æ0.30
1.5Æ0.84
4.3Æ0.65
4.3Æ0.49
4.9Æ0.73
2.3Æ0.72
7.6Æ0.73
4.9Æ0.58
26.0Æ0.83
4.4Æ0.47
3.3Æ0.61
6.6Æ0.26
2.4Æ0.55
3.7Æ0.30
77
53
32
41
170
29
49
20
37
73
32
286
78
91
106
74
434
Experimental Section
VRK1
ZAP70
11
5
413
Chemistry
[a] Affinity data are given as a percent of control (PoC). [b] Data represent
the meanÆSD; IC50 values were measured by testing 12 concentrations
of the test compound in the range from 1ꢄ10À04 m to 3ꢄ10À11 m (n=1)
in each kinase assay. [c] No valid IC50 value could be determined against
this kinase. [d] Ambiguous fitting.
General: All reagents and solvents were obtained from commercial
sources and were used without further purification unless other-
wise stated. Water used for preparing test samples was pure to
a resistivity of 18.2 MWcm (Milli-Q). DMSO used to prepare test
samples was purchased from Sigma–Aldrich (BioReagent).
[D6]DMSO and CDCl3 (99.8% D) were stored on molecular sieves
1
(3 ꢃ). H and 13C NMR spectra were recorded at 300 and 75 MHz,
mides 7 and 8. Notably, the enzyme inhibitory activity of 7, in
the low-micromolar range, is similar to its IC50 value obtained
from in vitro cell viability assays in the NCI-60 panel. This
strongly suggests that cyclosquaramide 7 can easily penetrate
cellular membranes without being affected by the usual degra-
dation pathways present in the cytoplasm.
respectively, at 238C unless otherwise specified. Chemicals shifts
(d) are reported in ppm referenced to the residual deuterium lock
solvents. 13C NMR spectra in D2O were referenced to 3-(trimethyl-
silyl)-1-propanesulfonic acid sodium salt. Coupling constants (J) are
given in Hz. IR spectra were recorded on an FTIR Bruker IFS66
spectrophotometer. HRMS data were obtained on a MicroMass Au-
tospec 3000 double-focusing magnetic sector mass spectrometer
operating at 3000 m/z at a cone voltage of 4 V. Samples were in-
troduced through an electrospray module (ESI) at 10À6 m in MeOH
at a rate of 20 mLminÀ1. The charge of the species corresponding
to an observed ion rate was deduced directly from the spacing of
the isotope peaks for masses <3000 m/z. Calibration was done
with PER standards, giving closest peaks to the molecular ions
under study. Analytical reversed-phase ion-pair HPLC was carried
out on an Atlantis T3 C18 5 mm 4.8ꢄ150 mm column, using mix-
tures of solution A (0.1m NaOAc and 0.01m sodium octanesulfo-
nate in Milli-Q H2O solution adjusted to pH 3.4 with acetic and
formic acids) and solution B (CH3CN) as mobile phase. Chromatog-
raphy was carried out at a flow rate of 1 mLminÀ1 over 35 min
starting from isocratic flux at 30% CH3CN (solution B) + 70% solu-
tion A for 20 min and then gradient from 30% CH3CN to 80%
CH3CN over 8 min. Luminescence was measured with a Hidex Cha-
meleon 425-104 plate reader using 96-well ELISA microplates.
To determine the mode of kinase inhibition by cyclosquara-
mide 7, an ATP-competitive binding assay was performed. The
kinase ABL1 was chosen for this experiment. For this purpose,
the IC50 values of the compound were determined in the ABL1
assay at four different ATP concentrations (0.1, 1, 3, and
10 mm). The values obtained, ranging from 0.4 to 0.7 mm, were
similar regardless of ATP concentration, clearly showing that in-
creasing concentrations of ATP do not lead to an increase in
IC50 value for the test compound in the ABL1 assay. On the
contrary, the IC50 values showed a slight decrease with increas-
ing amounts of ATP. Taken together, these results clearly indi-
cate that the test compound does not behave as a mere ATP
antagonist. For an ATP antagonist, an increase in IC50 value
with increasing ATP concentration would be expected. In addi-
tion, the very steep IC50 curves (absolute values of Hill slopes
@1) suggest that the test compound binds at more than one
site of the enzyme in a cooperative manner.
Compounds 4–8, 11, 12, and 17 were prepared as described previ-
ously.[21,22] Boc-methionine (2-(tert-butoxycarbonylamino)-4-methyl-
sulfanylbutanoic acid) 13 was prepared as described elsewhere.[34]
Macrocycle 9: Linear oligosquaramide 20, containing five squara-
mide units, was prepared as described elsewhere.[22] A solution of
&6
&
www.chemmedchem.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
ÝÝ
These are not the final page numbers!

Cyclosquaramides as Kinase Inhibitors
MED
HCl (3m, 1.2 mL, 3.6 mmol) was added to 20 (350 mg, 0.24 mmol)
in H2O (10 mL). The solution was warmed at 508C for 6 h. The mix-
ture was then cooled to room temperature and brought to pH 9–
10 by adding solid Na2CO3. The solvent was removed, and the resi-
due was suspended in MeOH (14 mL) and filtered. To this solution,
solid Na2CO3 (106 mg) and 12 (95 mg, 0.24 mmol) in MeOH (6 mL)
were added. The reaction was stirred at room temperature for
24 h. The solvent was then removed, and the crude was dissolved
in HCl (3m, 2 mL). NaOH (1m) was added to pH 9–10. The precipi-
tate was centrifuged, dried and extracted in CH2Cl2 (5 mL). Com-
pound 9 was obtained as a pale-yellow solid (288 mg, 77%):
1H NMR (300 MHz, [D6]DMSO): d=1.65 (br, 28H, CH2CH2CH2), 2.10
(s, 21H, NCH3), 2.30 (br, 28H, CH2N), 3.50 (br, 28H CH2NHsq),
7.43 ppm (br, 14H, NH); 13C NMR (75 MHz, D2O+HCl): d=28.0,
42.7, 43.8, 55.9, 171.7, 184.3 ppm; HRMS (ESI): m/z [M+Na]+ calcd
for C77H119N21NaO14: 1584.9138, found: 1584.9156.
[D6]DMSO): d=1.33 (s, 18H, C(CH3)3), 1.46 (m, 2H, CH2CH2S), 1.63
(br, 18H, NCH2CH2CH2N), 2.00 (s, 3H, SCH3), 2.08(s, 6H, NCH3), 2.08
(s, 6H, NCH3), 2.2–2.3 (m, 20H, (CH2)2NCH3, CH2NHCOCH, CH2S),
2.89 (m, 4H, CH2NHCOO), 3.47 (br, 14H, CH2NHsq), 4.63 (br, 1H,
NHCHCONH) 6.76 (br, 3H, CHCONHCH2 +NHCO), 7.50 ppm (br, 8H,
NHsq); 13C NMR (75 MHz, [D6]DMSO): d=14.6, 27.0, 28.2, 28.5, 38.2,
41.6, 54.1, 54.1, 54.2, 54.6, 77.3, 155.5, 167.8, 182.3 ppm; HRMS
(ESI): m/z [M+Na]+ calcd for C62H103N15O13SNa+: 1320.747, found:
1320.8105.
Macrocycle 1: A solution of HCl (3m, 1 mL, 3 mmol) was added to
15 (0.314 g, 0.24 mmol) in H2O (15 mL). The solution was warmed
at 508C for 12 h. The mixture was then cooled to room tempera-
ture and brought to pH 9–10 by adding solid Na2CO3. The solvent
was removed, and the residue was suspended in EtOH (10 mL) and
filtered. To this solution, solid Na2CO3 (106 mg) and diethyl squa-
rate (0.045 g, 0.26 mmol) in EtOH (1 mL) were added. The reaction
was stirred at room temperature for 24 h. The solvent was then re-
moved, and the crude was dissolved in HCl (3m, 2 mL). NaOH (1m)
was added until pH 9–10. The precipitate was centrifuged, dried
and extracted in CH2Cl2 (5 mL). Compound 1 was obtained as
(S)-tert-Butyl
thio)butanamido)propyl)carbamate (14): Compound 13 (1.0 g,
4.01 mmol), N-tert-butoxycarbonyl-1,3-diaminopropane (0.7 g,
(3-(2-((1-(tert-butoxycarbonyl)amino)-4-(methyl-
4.02 mmol), and DMAP (0.05 g, 0.41 mmol) in dry CH2Cl2 (30 mL)
were added dropwise to a stirred solution of DCC (1.076 g,
5.2 mmol) in CH2Cl2 (10 mL) at 08C under inert (Ar) gas. The mix-
ture was allowed to stand at 08C for 2 h and an additional 12 h at
room temperature. The crude mixture was filtered. The solvent was
removed, and the residue was suspended in Et2O (10 mL) and fil-
tered. The addition of hexane to the solution afforded 14 as
1
a pale-yellow solid (0.1 g, 35%): H NMR (300 MHz, [D6]DMSO): d=
1.62 (br, 18H, CH2CH2CH2 +1H CHCHaHbCH2S), 1.99 (br, 3H, SCH3 +
1HCHCHaHbCH2S), 2.07 (s, 12H, NCH3), 2.30 (br, 16H, CH2N), 3.11
(br, 2H, CH2CH2S), 3.48 (br, 18H, CH2NH(sq)+2HCH2NHCO), 4.59
(br, 1H, NHCHCONH), 8,12 (br, 10H, NH) 8.2 ppm (br, 1H, NHCO);
13C NMR (75 MHz, [D6]DMSO): d=17.7, 31.6, 32.0, 33.9, 39.0, 44.6,
57.2, 58.0, 171.0, 185.3 ppm; HRMS (ESI): m/z [M+Na]+ calcd for
C56H85N15NaO11S: 1198.6166, found: 1198.6171.
1
a white solid (1.138 g, 70%): H NMR (300 MHz, CDCl3): d=1.44 (s,
18H, C(CH3)3), 1.62 (m, 2H, NHCH2CH2CH2), 1.92 (m, 1H,
CHCH2CH2S), 2.11 (m, 1H, CHCH2CH2S), 2.11 (s, 3H, SCH3), 2.55 (m,
2H, CH2CH2NHCOO), 3.15 (m, 2H, CH2CH2S), 3.31 (m, 2H,
CONHCH2CH2), 4.20 (m, 1H, NHCHCONH), 4.90 (br, 1H, NHCOO), 5.2
(br, 1H, NHCOO), 6.7 ppm (br, 1H, CONH); 13C NMR (75 MHz,
CDCl3): d=15.4, 15.5, 28.5, 28.6, 30.4, 36.1, 37.1, 66.0, 77.4, 156.6,
157.2, 171.9 ppm; HRMS (ESI): m/z [M+Na]+ calcd for
C18H35N3O5SNa: 428.2195, found: 428.2198.
Macrocycle 2: Compound 2 was prepared and purified as de-
scribed above for 1 by starting from 16 (0.4 g, 0.31 mmol) in
MeOH and diester 12 (0.133 g, 0.34 mmol). Purification of the
crude product by washing with THF (2ꢄ5 mL) and acetone (2ꢄ
5 mL) afforded 2 as a pale-yellow solid (0.176 g, 41%). 1H NMR
(300 MHz,
[D6]DMSO):
d=1.63
(br,
22H
CH2CH2CH2
+1HCHCHaHbCH2S), 1.99 (s, 3H, SCH3 +1HCHCHaHbCH2S), 2.09 (s,
15H, NCH3), 2.29 (br, 20H, CH2N), 3.12 (br, 2H, CH2CH2S), 3.48 (br,
22H, CH2NH(sq) +2HCH2NHCO), 4.61 (br, 1H, NHCHCONH),
7.42 ppm (br, 13H, NH); 13C NMR (75 MHz, D2O-HCl): d=16.9, 28.0,
31.8, 39.2, 42.7, 43.8, 55.9, 170.7, 175.3 184.3 ppm; HRMS (ESI): m/z
[M+H]+ calcd for C67H103N18O13S: 1399.7667, found: 1399.7673.
Oligosquaramide 15: Compound 14 (1.117 g, 2.05 mmol) was sus-
pended in a solvent mixture of H2O (30 mL) and MeOH (20 mL);
HCl (3m, 6.6 mL, 19.8 mmol) was then added until complete disso-
lution of 13. The solution was stirred for 2 h at 508C and an addi-
tional 12 h at 408C. The cooled solution was then carefully brought
to pH 9–10 with solid Na2CO3. The solvent was removed, and the
residue was suspended in EtOH (10 mL) and filtered. The resulting
solution was added dropwise to a suspension of 11 (1.59 g,
4.3 mmol) and Na2CO3 (2.12 g) in EtOH (20 mL). The reaction was
stirred at room temperature for 24 h, then the solvent was re-
moved, and the crude was suspended in CH2Cl2 (20 mL), washed
with H2O (2ꢄ10 mL), saturated NaCl(aq) (10 mL), and dried with
Na2SO4. The solvent was removed, and the solid obtained was ex-
tracted in Et2O (3ꢄ20 mL) to furnish 15 as a pale-yellow solid
Macrocycle 3: Compound 3 was prepared in 30% yield as de-
scribed for compound 7 from the trimeric aminosquaramide and
diester 17. 1H NMR (300 MHz, [D6]DMSO): d=1.27 (br, 6H,
NHCH2CH2(CH2)3), 1.48 (br, 4H, NHCH2CH2(CH2)3), 1.64 (br, 16H,
NCH2CH2CH2N), 2.09 (s, 12H, NCH3), 2.29 (br, 16H, CH2NCH2), 3.48
(br, 20H, CH2NH(sq)), 7.47 ppm (br, 10H, NH); 13C NMR (75 MHz,
D2O-HCl): d=27.9, 32.6, 42.8, 43.9, 47.1, 55.9, 170.3, 170.7, 183.4,
184.1 ppm; HRMS (ESI): m/z [M+H]+ calcd for C55H85N14O10:
1101.6568, found: 1101.6580.
1
(1.473 g, 84%): H NMR (300 MHz, CDCl3): d=1.42 (s, 18H, C(CH3)3),
1.64 (br, 4H, NCH2CH2CH2N), 1.83 (br, 6H NCH2CH2CH2N), 2.08 (s,
5H, CH2CH2SMe+SCH3), 2.20 (s, 6H, NCH3), 2.40–2.56 (br, 8H,
CH2NCH3, CHCONHCH2), 3.15 (br, 8H, CH2NHCO,CH2S),3.71 (br, 6H,
CH2NHCOO) 4.70 (br, 1H, NHCHCONH), 4.9–5.4 (br, 3H, NHCOO),
7.9 ppm (br, 4H, NHsq, NHCO); 13C NMR (75 MHz, CDCl3): d=15.7,
27.3, 28.6, 30.1, 39.2, 41.9, 43.3, 55.2, 77.3, 156.5, 167.6, 182.6 ppm;
HRMS (ESI): m/z [M+H]+ calcd for C40H70N9O9S: 852.5017, found:
852.5017.
Oligosquaramide 18: Compound 18 was prepared as described
above for compound 15 from N1-(3-aminopropyl)-N1-benzylpro-
pane-1,3-diamine (445 mg, 2.01 mmol) and compound 11 (1.78 g,
1
4,82 mmol) to give 18 as a pale-yellow solid (1.32 g, 74%): H NMR
(300 MHz, [D6]DMSO): d=1.36 (s, 18H, C(CH3)3), 1.49 (m, 4H,
NCH2CH2CH2N), 1.66 (m, 8H, NCH2CH2CH2N), 2.10 (s, 6H, CH3),
2,27(m, 8H, NCH2CH2CH2N), 2.42 (m, 4H, NCH2CH2CH2N), 2.91 (m,
4H, CH2NHCOO), 3.52 (m, 10H, NCH2Ar), 6.76 (br, 2H, CH2NHCOO),
7.10–7.60 ppm (m, 5H, Ar+4HNH); 13C NMR (75 MHz, [D6]DMSO):
d=27.7, 29.0, 39.0, 42.3, 42.4, 50.9, 55.0, 55.3, 58.4, 78.1, 127.5,
128.8, 129.4, 140.1, 156.4, 168.7, 183.1 ppm; HRMS (ESI): m/z [M+
Na]+ calcd for C45H73N9NaO8+: 890.5474, found: 890.5441.
Oligosquaramide 16: Compound 16 was prepared and purified as
described above for 15 by starting from 15 (1.0 g, 1,17 mmol) and
11 (0.91 g, 2.46 mmol) and using 8 equiv HCl. The desired product
1
16 was obtained as a white solid (0.758 g, 50%): H NMR (300 MHz,
ChemMedChem 0000, 00, 1 – 10
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
&
7
&
ÞÞ
These are not the final page numbers!

C. Rotger, A. Costa, et al.
MED
Oligosquaramide 19: Compound 19 was prepared and purified as
described above for 16 by starting from 18 (486 mg, 0.60 mmol)
and 11 (454 mg, 1.23 mmol). The desired product was obtained as
a white solid (477 mg, 60%). 1H NMR (300 MHz, [D6]DMSO): d=
1.35 (s, 18H, C(CH3)3), 1.48 (m, 4H, NCH2CH2CH2N), 1.64 (m, 16H,
NCH2CH2CH2N), 2.10 (s, 12H, CH3), 2,24 (br, 16H, NCH2CH2CH2N),
2.40 (m, 4H, NCH2CH2CH2N), 2.90 (m, 4H, CH2NHCOO), 3.49 (br, 2H,
NCH2Ar+16HCH2NH(sq)), 6.76 (br, 2H, CH2NHCOO), 7.00–7.80 ppm
(m, 5H Ar+8HNH); 13C NMR (75 MHz, [D6]DMSO): d=27.2, 28.7,
37.9, 42.0, 42.1, 50.4, 54.5, 54.9, 58.0, 78.2, 78.5, 127.2, 128.5, 129.2,
139.5, 156.3, 168.1, 182.5 ppm; HRMS (ESI): m/z [M+Na]+ calcd for
C67H107N15NaO12+: 1336.8116, found: 1336.8148.
Multicellular tumor spheroid formation assays: To monitor the for-
mation of multicellular tumor spheroids in culture, 4ꢄ105 U87MG
cells were plated in six-well plates and treated as indicated in the
figure legends. After 4–6 days, cells were stained with 0.5% (w/v)
crystal violet in 70% EtOH, and the number of multicellular tumor
spheroids from representative fields (>10) were counted under
a light microscope.
Flow cytometry: Cell-cycle analysis was performed using propidium
iodide staining. Briefly, Jeko-1 and JVM-2 cells were treated with
cyclosquaramide 7 for 24 h, and were then washed in PBS and
fixed in 90% EtOH. Fixed cells were washed twice in PBS and
stained in 50 mm propidium iodide containing 5 mgmLÀ1 DNase-
free RNase for 1 h, then processed by flow cytometry using a FACS-
can (Coulter Epics XL-MSL, Beckman Coulter, Fullerton, CA, USA)
and analyzed with winMDI software.
Macrocycle 10: Compound 10 was prepared as described above
for compound 7 by starting from oligosquaramide 19 and diethyl
squarate. In a typical procedure, 19 (477 mg, 0.363 mmol) was dis-
solved in H2O (15 mL) and HCl (3m, 1.1 mL, 3.3 mmol) and stirred
for 5 h at 508C. The resulting solution was cooled to room temper-
ature, and solid Na2CO3 was carefully added to pH 9. The solvent
was evaporated, and the crude residue was dissolved in DMSO
(2 mL) and MeOH (40 mL). The solution was filtered to eliminate
salts prior to the addition of diethyl squarate (54 mL, 0.365 mmol)
in MeOH (10 mL). The reaction mixture was stirred at room temper-
ature for 72 h. The solvent was removed, and the crude was dis-
solved in aqueous Na2CO3 (5%, 20 mL) and extracted with CHCl3
(4ꢄ15 mL) and CHCl3/tBuOH 50:50 v/v (2ꢄ15 mL). The organic
layers were dried over anhydrous Na2SO4, and the solvent was re-
moved. The solid residue was crystallized from CH2Cl2/Et2O (30:70)
Kinase inhibition assays: These assays were performed by ProQinase
GMBH, Freiburg (Germany), according to the procedure described
by ProQinase.[35] A radiometric protein kinase assay (PanQinaseActi-
vityAssay) was used for measuring the kinase activity of the 25 pro-
tein kinases. All kinase assays were performed in 96-well Flash-
Plates from PerkinElmer (Boston, MA, USA) in a 50 mL reaction
volume. The reaction cocktail was pipetted in four steps in the fol-
lowing order: 15 mL ATP solution (in H2O), 20 mL assay buffer (see
below), 5 mL test sample in 10% DMSO, and 10 mL enzyme/sub-
strate mixture (in storage buffer). The assay for all enzymes con-
tained 60 mm HEPES·NaOH (pH 7.5), 3 mm MgCl2, 3 mm MnCl2,
3 mm Na3VO4, 1.2 mm DTT, 50 mgmLÀ1 PEG 20000, 1 mm [g-33P]ATP
(~6ꢄ105 cpm per well), protein kinase (variable amounts; see
table S1 in the Supporting Information), and substrate (variable
amounts; see table S1).
1
to yield 10 as a pale-yellow solid (303 mg, 70%). H NMR (300 MHz,
[D6]DMSO): d=1.67 (br, 20H, CH2), 2.14 (br, 12H, NCH3), 2.38 (br,
20H, CH2), 3.03 (br, 2H, CH2), 3.50 (br, 20H, CH2), 8.2–7.2 ppm (br,
15H, NH+C6H5); 13C NMR (75 MHz, [D6]DMSO): d=27.8, 42.7, 44.0,
52.5, 55.9, 131.4, 131.9, 132.6, 133.4, 170.3, 182.9 ppm; HRMS (ESI):
m/z [M+H]+ calcd for C61H90N15O10: 1192.6995, found: 1192.6969.
Evaluation of the mode of ABL1 inhibition: A radiometric filter-bind-
ing assay was used for measuring the kinase activity under various
conditions. All kinase assays were performed in 96-well polypropy-
lene microtiter plates in a 50 mL reaction volume. The reaction
cocktail was pipetted in five steps in the following order: 20 mL
assay buffer (standard buffer), 5 mL test compound (in 10% DMSO),
10 mL enzyme solution, 10 mL substrate solution, and 5 mL ATP solu-
tion (in H2O). The assay contained 60 mm HEPES·NaOH (pH 7.5),
3 mm MgCl2, 3 mm MnCl2, 3 mm Na3VO4, 1.2 mm DTT, 50 mgmLÀ1
PEG 20000, and various amounts of [g-33P]ATP (~1.5ꢄ106 cpm per
well).
Biology
Cell culture: The human MCL cell lines Jeko-1, JVM-2, Granta-519,
Rec-1, and Z-138 were maintained in RPMI 1640 medium. Human
glioblastoma cell lines T98G, U373, U87MG, and LN229, human os-
teosarcoma U2OS cells, and human colorectal cancer SW620 cells
were maintained in DMEM. All media were supplemented with
10% heat-inactivated fetal bovine serum and 100 UmLÀ1 penicillin/
streptomycin. All cell lines were grown under humidified air con-
taining 5% CO2 at 378C.
Acknowledgements
In vitro viability assays with compounds 1–10: The number of viable
cells in culture was determined based on quantification of ATP,
which signals the presence of metabolically active cells, using the
Cell Titer-Glo luminescent assay kit (Promega, Madison, WI, USA)
by following the manufacturer’s instructions. Briefly, cells (4ꢄ
105 cellsmLÀ1) were treated with the compound under assay (1–9)
for 48 h followed by the addition of Cell Titer-Glo Reagent. Twelve
final concentrations were tested: 200, 50, 20, 8, 3, 1, 0.3, 0.1, 0.05,
and 0.01 mm. Moreover, DMSO was added as a negative control
(0.01% v/v). Luminescence was detected using a multi-well scan-
ning spectrophotometer (Plate Chameleon, Hidex, Finland). Cell vi-
ability is represented as a percentage relative to vehicle-treated
cells, and data are the mean of three independent determinations
performed in duplicate. IC50 is defined as the compound concen-
tration required to produce 50% inhibition of growth relative to
vehicle-treated control. IC50 values were calculated from sigmoidal
analysis of dose–response curves using GraphPad Prism v.5 for
MacOS (GraphPad Software Inc., La Jolla, CA, USA).
We are grateful to Joan Seoane (Institut de Recerca Hospital Vall
d’Hebron, Barcelona), Dolors Colomer (Hospital Clꢀnic, Barcelona),
and Beatriz Martꢀnez (CNIO, Madrid) for the generous gift of cell
lines. This research is supported by the Ministry of Science and In-
novation (MICINN) of Spain (grants CTQ2008-00841/BQU and
CTQ2011-27152), MICINN Consolider-Ingenio (grant CSD2010-
00065), and CAIB (grant 23/2011). We also acknowledge an AERD
(2008) grant of CAIB. P.V. is a Ramꢂn y Cajal Fellow (MICINN,
Spain).
Keywords: antitumor agents · biological activity · inhibitors ·
macrocycles · squaramides
[1] Throughout this paper the term “squaramide” is used to designate 3,4-
diamino derivatives of 3,4-dihydroxycyclobut-3-ene-1,2-dione (squaric
&8
&
www.chemmedchem.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
ÝÝ
These are not the final page numbers!

Cyclosquaramides as Kinase Inhibitors
MED
acid). We coined the term “cyclosquaramide” to designate cyclic oligo-
meric aminoalkyl-squaramide compounds.
[2] D. QuiÇonero, C. Garau, A. Frontera, P. Ballester, A. Costa, P. M. Deyꢅ,
Chem. Eur. J. 2002, 8, 433–438.
[3] C. Rotger, B. Soberats, D. QuiÇonero, A. Frontera, P. Ballester, J. Benet-
Buchholz, P. M. Deyꢅ, A. Costa, Eur. J. Org. Chem. 2008, 1864–1868.
[4] S. Tomꢅs, R. Prohens, G. Deslongchamps, P. Ballester, A. Costa, Angew.
Chem. 1999, 111, 2346–2349; Angew. Chem. Int. Ed. 1999, 38, 2208–
2211.
[5] J. P. Malerich, K. Hagihara, V. H. Rawal, J. Am. Chem. Soc. 2008, 130,
14416–14417.
[6] R. Prohens, M. C. Rotger, M. N. PiÇa, P. M. Deyꢅ, J. Morey, P. Ballester, A.
Costa, Tetrahedron Lett. 2001, 42, 4933–4936.
[17] J. C. Pickens, D. D. Mitchell, J. Liu, X. Tan, Z. Zhang, C. L. M. J. Verlinde,
W. G. J. Hol, W. E. Fan, Chem. Biol. 2004, 11, 1205–1215.
[18] A. Crew, A. H. Li, L. Qiu, A. Castelhano, H. Dong, A. Smith, L. Tardibono,
T. Zhang (OSI Pharmaceuticals Inc.), WO/2006/034111, 2006.
[19] J. W. Janetka, S. Ashwell, Expert Opin. Ther. Pat. 2009, 19, 165–197.
[20] The difference between conformers in amides is 0.5–2.5 kcalmolÀ1, and
the free-energy barrier for cis–trans interconversion is 16–22 kcal
molÀ1. See: C. Dugave, L. Demange, Chem. Rev. 2003, 103, 2475–2532.
[21] M. C. Rotger, M. N. PiÇa, A. Frontera, G. Martorell, P. Ballester, P. M. Deyꢅ,
A. Costa, J. Org. Chem. 2004, 69, 2302–2308.
[22] C. Rotger, M. N. PiÇa, M. Vega, P. Ballester, P. M. Deyꢅ, A. Costa, Angew.
Chem. 2006, 118, 6998–7002; Angew. Chem. Int. Ed. 2006, 45, 6844–
6848.
[7] K. Sato, R. Tawarada, K. Seio, M. Sekine, Eur. J. Org. Chem. 2004, 2142–
2150.
[23] L. A. Wessjohann, E. Ruijter, D. Garcia-Rivera, W. Brandt, Mol. Diversity
2005, 9, 171–186.
[8] K. Sato, K. Seio, K. Sekine, J. Am. Chem. Soc. 2002, 124, 12715–12724.
[9] J. A. Butera, M. M. Antane, S. A. Antane, T. M. Argentieri, C. Freeden, R. F.
Graceffa, B. H. Hirth, D. Jenkins, J. R. Lennox, E. Matelan, N. W. Norton,
D. Quagliato, J. H. Sheldon, W. Spinelli, D. Warga, A. Wojdan, M. Woods,
J. Med. Chem. 2000, 43, 1187–1202.
[10] A. M. Gilbert, M. M. Antane, T. M. Argentieri, J. A. Butera, G. D. Francisco,
C. Freeden, E. G. Gundersen, R. F. Graceffa, D. Herbst, B. H. Hirth, J. R.
Lennox, G. McFarlane, N. W. Norton, D. Quagliato, J. H. Sheldon, D.
Warga, A. Wojdan, M. Woods, J. Med. Chem. 2000, 43, 1203–1214.
[11] C. W. Lee, H. Cao, K. Ichiyama, T. M. Rana, Bioorg. Med. Chem. Lett. 2005,
15, 4243–4246.
[12] K. Urbahns, M. Hꢆrter, M. Albers, D. Schmidt, B. Stelte-Ludwig, U. Brꢇg-
gemeier, A. Vaupel, J. Keldenich, K. Lustig, H. Tsujishita, C. Gerdes,
Bioorg. Med. Chem. Lett. 2007, 17, 6151–6154.
[13] Y. F. Xie, K. Lake, K. Ligsay, M. Komandla, I. Sircar, G. Nagarajan, J. Li, K.
Xu, J. Parise, L. Schneider, D. Huang, J. Liu, K. Dines, N. Sakurai, M. Bar-
bosa, R. Jack, Bioorg. Med. Chem. Lett. 2007, 17, 3367–3372.
[14] B. W. McCleland, R. S. Davis, M. R. Palovich, K. L. Widdowson, M. L.
Werner, M. Burman, J. J. Foley, D. B. Schmidt, H. M. Sarau, M. Rogers,
Bioorg. Med. Chem. Lett. 2007, 17, 1713–1717.
[24] R. M. Owen, C. B. Carlson, P. Mowery, J. Xu, E. Fasella, L. L. Kiessling,
ChemBioChem 2007, 8, 68–82.
[25] A. B. Pomilio, M. E. Battista, A. A. Vitale, Curr. Org. Chem. 2006, 10,
2075–2121.
[26] S. C. Brauns, S. P. Milne, R. Naudꢈ, M. Van de Venter, Anticancer Res.
2004, 24, 1713–1720.
[27] Y. L. Janin, Amino Acids 2003, 25, 1–40.
[28] A. Obrador-Hevia, S. Fernꢀndez de Mattos, P. Villalonga, J. Rodrꢉguez,
Blood Rev. 2009, 23, 205–216.
[29] S. S. Ren, E. J. Lien, Curr. Pharm. Des. 2004, 10, 1399–1415.
[30] E. M. Driggers, S. P. Hale, J. Lee, N. K. Terrett, Nat. Rev. Drug Discovery
2008, 7, 608–624.
[31] M. R. Boyd, K. D. Paull, Drug Dev. Res. 1995, 34, 91–109.
[32] A. Kolchinsky, I. B. Roninson, Anticancer Res. 1997, 17, 3321–3327.
[33] P. M. LoRusso, J. P. Eder, Exp. Op. Inv. Drugs 2008, 17, 1013–1028.
[34] J. Zhang, P. L. Yang, N. S. Gray, Nat. Rev. Cancer 2009, 9, 28–39.
[35] ProQinase GmbH, http://www.proqinase.com/ (accessed June 18, 2012).
[15] E. A. Merritt, Z. Zhang, J. C. Pickens, M. Ahn, W. G. J. Hol, E. Fan, J. Am.
Chem. Soc. 2002, 124, 8818–8824.
[16] E. Fan, Z. Zhang, W. E. Minke, Z. Hou, C. L. M. J. Verlinde, W. G. J. Hol, J.
Am. Chem. Soc. 2000, 122, 2663–2664.
Received: March 21, 2012
Revised: June 15, 2012
Published online on && &&, 0000
ChemMedChem 0000, 00, 1 – 10
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
&
9
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
P. Villalonga, S. Fernꢁndez de Mattos,
G. Ramis, A. Obrador-Hevia, A. Sampedro,
C. Rotger,* A. Costa*
It’s hip to be square: A new series of
oligosquaramide macrocycles were syn-
thesized and evaluated as anticancer
agents. Sensitivity to cyclosquaramides
clearly depends on cell type, indicating
a degree of biological selectivity. The
macrocyclic structure with minimal mo-
lecular size plays a key role in the ob-
served antitumor activity, and the anti-
proliferative activity is related to the ef-
fective inhibition of several important
kinases.
&& – &&
Cyclosquaramides as Kinase Inhibitors
with Anticancer Activity
&10
&
www.chemmedchem.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 10
ÝÝ
These are not the final page numbers!