Synthesis and antitumor activity of mechercharmycin A analogues

Article abstract of DOI:10.1021/jm800513w

Several analogues of the cytotoxic thiopeptide IB-01211 or mechercharmycin A (1) have been synthesized. The cytotoxicity of 1 and the synthesized analogues were evaluated against a panel of three human tumor cell lines. Thiopeptide 1 and the most active d

Full text of DOI:10.1021/jm800513w

5722
J. Med. Chem. 2008, 51, 5722–5730
Synthesis and Antitumor Activity of Mechercharmycin A Analogues
,†,§
Delia Herna´ndez,† Marta Altuna,† Carmen Cuevas,# Rosa Aligue´,| Fernando Albericio,*,†,‡ and Mercedes Alvarez*
´
Institute for Research in Biomedicine, Barcelona Science Park-UniVersity of Barcelona, Baldiri Reixac 10,
E-08028 Barcelona, Spain, CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park,
Baldiri Reixac 10, E-08028 Barcelona, Spain, Pharma Mar, S.A., AVda de los Reyes 1, E-28770 Colmenar Viejo, Madrid, Spain, Department
of Cell Biology, Faculty of Medicine, UniVersity of Barcelona, CasanoVa 143, E-08036 Barcelona, Spain, and Department of Organic
Chemistry and Laboratory of Organic Chemistry, Faculty of Pharmacy, UniVersity of Barcelona, E-08028 Barcelona, Spain
ReceiVed May 5, 2008
Several analogues of the cytotoxic thiopeptide IB-01211 or mechercharmycin A (1) have been synthesized.
The cytotoxicity of 1 and the synthesized analogues were evaluated against a panel of three human tumor
cell lines. Thiopeptide 1 and the most active derivatives 2 and 3c were chosen for further studies on effects
on cell cycle progression and induction of apoptosis. Interestingly, the inhibition of cell division and activation
of a programmed cell death by apoptosis were detected.
Introduction
exocyclic double bond via elimination (2) or conjugation of the
hydrate form 3 with acids, PEGa residues, or amino acids
(3a-f).
Natural products are a rich source for drug discovery and
are challenging synthetic targets; hence, they often inspire new
synthetic methods.1 Thiazoles and oxazoles, as well as their
reduced variants, are common structural features of many
biologically important natural products.2 Only few concatenated
azoles containing substituents on the azole rings have been
described. These comprise the methylated bisoxazoles (-)-
muscoride A3 and leucamide A,4 the cyclic octaazole telom-
estatin,5 which features two oxazole rings with methyl substit-
uents at position 5, and finally, the diazonamides,6 which have
a complex structure containing a bis-oxazole. IB-01211 or
mechercharmycin A (1) is a cyclic thiopeptide7 containing a
phenylpentaazole system, a D-allo-Ile-L-Val dipeptide, and an
exocyclic methylidene. The synthesis of 1 was recently described
by our group.8 Thiopeptide 1 is closely related in structure to
the potent telomerase inhibitor telomestatin5 and YM-216391.9
We sought to prepare analogues of 1 with improved solubility
and activity. The first structural modifications comprised
substitution on the pentaazole system and modifications of the
exocyclic methylidene (Figure 1). We envisaged that introduc-
tion of one (1a, b) or two (1c) methyl groups could increase
the solubility of the parent compound and allow a conforma-
tional change. The second variation consisted of modifying the
Results and Discussion
Chemistry. Compounds 1a-c, 2, and 3a-f were synthesized
using basically the same strategy that we described for the total
synthesis of 1.8a This is a convergent method based on
macrocyclization with simultaneous formation of the thiazole
ring from a peptide-heterocycle. Having previously reported the
difficulty of handling the alcohol 3,8 we used different conditions
for the macrocyclization of the intermediates leading to 2 and
the esters 3a-e than for those leading to 1a-c, which feature
an exocyclic methylidene. Thus, whereas 3a-e were all
prepared from 3 via experimental conditions that avoid dehydra-
tion, 1a-c were obtained through concomitant macrocyclization
and formation of the thiazole and the double bond.
The syntheses were built around three synthetic intermediates:
the bis-oxazoles 4a-c, the phenyl-bis-oxazoles 5a and 5b, and
the dipeptide 6 (Scheme 1). Compounds 4a and 5a have
previously been synthesized8 by a process based on cyclization
of Ser and Ph-Ser peptides to give an oxazoline, followed by
further oxidation to afford the oxazole. This method was used
to prepare oxazoles 4b, 4c, and 5b, using Gly- and Thr-
containing peptides, as well as Ser and Ph-Ser, as starting
materials (see Experimental Section). The peptide-heterocycles
7a and 7b were obtained in excellent yields from the peptide 6
and compounds 5a, 5b, respectively, using EDC ·HCl (1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and
HOBt (1-hydroxybenzotriazole) as coupling agents. The tet-
raoxazole peptides 9 were obtained from the corresponding free
acid of 710 and the appropriate amine 8 under the conditions
described above. Compounds 9a-e feature three key functional
groups: a thioamide, an acetal-protected R-bromoketone, and a
tert-butoxymethyl group (R ) CH2OtBu for 9a-d). Simulta-
neous elimination of the OtBu protecting group and the
dimethylacetal of 9 using formic acid under reflux afforded the
free alcohol plus the R-bromoketone, which was ready for
macrocyclization via thiazole formation. Macrocyclization was
achieved by refluxing a dilute ethanolic solution of the crude
material for 48 h.11 For 1a-c (Scheme 1, left), this reaction
was followed by dehydration of the crude material using mesyl
chloride and triethylamine (TEA) at low temperature. The last
* To whom correspondence should be addressed. For F.A.: phone, (+34)
93403 7088; fax, (+34) 93 403 7126; e-mail, fernando.albericio@irbbarcelona.
org. For M.A.: phone, (+34) 93403 7086; fax, (+34) 93403 7126; e-mail,
mercedes.alvarez@irbbarcelona.org.
†
Barcelona Science Park-University of Barcelona and CIBER-BBN,
Networking Centre on Bioengineering, Biomaterials and Nanomedicine.
#
Pharma Mar, S.A.
Department of Cell Biology, Faculty of Pharmacy, University of
|
Barcelona.
‡
Department of Organic Chemistry, University of Barcelona.
Laboratory of Organic Chemistry, Faculty of Pharmacy, University of
§
Barcelona.
a Abreviations: A-549, human lung cancer cells; DAST, diethylamino-
sulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DIEA, diiso-
propylethylamine; DMAP, 4-dimethylaminopyridine; DMF, dimethylfor-
mamide; DMSO, dimethyl sulfoxide; EDC·HCl, 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride; FAM-DEVD-FMK, carboxyfluorescein
derivative of fluoromethyl ketone; HOBt, 1-hydroxybenzotriazole; HT-29,
human colon cancer cells; HTB, human colorectal adenocarcinoma cell line;
LR, Lawesson’s reagent; MsCl, mesyl chloride, NSCL, non-small-cell lung;
MDA-MB-231, human breast adenocarcinoma cancer cells; PBS, phosphate
buffered saline; PEG, poly(ethylene glycol); TEA, triethylamine; TFA,
trifluoroacetic acid; THF, tetrahydrofuran.
10.1021/jm800513w CCC: $40.75
2008 American Chemical Society
Published on Web 09/03/2008

Cyclic Thiopeptide Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5723
Figure 1. Structures of thiopeptide 1 and analogues 1a-c, 2, and 3a-f.
step after the macrocyclization was avoided when 9e was used
to give compound 2. By use of 9a, this route gave, after
deprotection and macrocyclization, alcohol 3, which was directly
acylated to give 3a-e. Acetate 3a was obtained using acetic
anhydride in THF at room temperature. Compounds 3b-e were
prepared by reaction of the appropriate acid with the alcohol 3
using EDC·HCl and 4-dimethylaminopyridine (DMAP) as
condensation agents. The protection initially used for Arg was
NR-Boc, bis-Nγ,γ′-Alloc, which allowed isolation of 3d in 16%
yield under the conditions described above. Elimination12 of
the Alloc protecting group gave a crude product in which NR-
Boc-3f only corresponded to 3% (as detected by HPLC-MS).
The deprotection gave better yield using tri-Boc-protected Arg.
Simultaneous elimination of the three protecting groups of 3e
using trifluoroacetic acid (TFA) diluted in CH2Cl2 allowed the
isolation with a 17% yield of purified 3f.
A-549 and HT-29) and 1c (all three lines) and by 2 orders
of magnitude for 1a (all three lines) and 1b (MDA-MB-231).
The greatest cytotoxicity is found in derivatives in which
the exocyclic double bond was eliminated (2) or substituted
by a PEG-carboxymethyl (3b and 3c). Acetate derivative 3a
maintains a modest growth inhibition at micromolar concen-
trations in the three lines; however, acylation with an Arg
residue leads to total loss of activity. Interestingly, substitu-
tion of the acetyl residue by a methoxyethoxyacetyl in 3b
increases cytotoxicity to A-549; likewise, the longer poly-
etheracetyl of 3c produces the same effect in all three tumor
cell lines. Compound 2 is a demethylidene analogue of 1
and also a constitutional isomer of YM-216391. Compound
2 and YM-216391 differ only in the position of the thiazole
ring inside the pentaazole structure of the macrocycle.13 The
open ring precursors 9a-e are generally inactive; only 9c is
moderately active against two tumor cell lines at micromolar
concentrations. The azole analogues14 of 1 recently obtained
by our group, which do not contain the central thiazole, were
also inactive. These results demonstrate the importance of
the thiazole for the activity of these macrocyclic compounds.
Biological Results
The cytotoxicity of the thiopeptide 1 analogues was evaluated
against a panel of three human tumor cell lines: A-549 lung
carcinoma NSCL, HT-29 colon carcinoma, and MDA-MB-231
breast adenocarcinoma.
A conventional colorimetric assay was used to estimate values
of GI50 (defined here as the drug concentration that causes 50%
of cell growth inhibition after 72 h of continuous exposure to
the test molecule). Thiopeptide 1 was included for comparison.
The results are shown in Table 1.
All the cyclic analogues, except 3f, are active against
human tumor cell lines but to a lesser extent compared to 1.
Introduction of one or two methyl substituents (1a-c)
diminishes the GI50 by 1 order of magnitude for 1b (lines
Thiopeptide 1 and the most active analogues 2 and 3c were
then selected for further biological evaluation.
Effects of 1, 2, and 3c on Cell Cycle Progression of
HT-29, A-549, and MDA-MB-231 Cells. Thiopeptide 1 is
closely related in structure to potent telomerase inhibitors
such as telomestatin. Telomerase generates telomere repeats,
specialized structures located at the end of eukaryotic
chromosomes. Telomeres are important for maintenance of
genomic stability and integrity during cell proliferation.
Thiopeptide 1 and their analogues 2 and 3c were tested for

5724 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Scheme 1. Synthesis of Thiopeptide 1 Analogues
Hernández et al.
effects on cell cycle progression in HT-29, A-549, and MDA-
MB-231 tumor cells. The cells were treated with each
compound (see Experimental Section) and then analyzed by
flow cytometry to determine if the cell cycle had been arrested
at a specific phase. A control set of untreated cells was also
used.

Cyclic Thiopeptide Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5725
Table 1. In Vitro Cytotoxicity of 1 and Its Open-Chain and Cyclic
cell cycle progression. We observed that 1 and compound 2
caused cells to accumulate mainly in G2 phase, which could
activate the G2-checkpoint activation. In contrast, 3c, though
less active than 1, provoked major S phase arrest in all cell
lines, indicating that it has potent biological activity related to
DNA replication. Moreover, the effects observed consecutively
to the treatment of cells with 3c resemble those expected for a
telomerase inhibitor.
Perturbation of the cell cycle induces activation of apoptosis
and leads to cell cycle arrest. Different cancer cell lines treated
with 1, 2, or 3c specifically underwent apoptosis, thereby
indicating that these compounds do not act by some general
toxicity mechanism. These compounds inhibit cell division and
activate programmed cell death by apoptosis.
Peptide Analoguesa
cytotoxicity (GI50, µM)
compd
A-549
HT-29
MDA-MB-231
j1
1a
1b
1c
2
3a
3b
3c
3f
0.03
1.66
0.54
0.31
0.17
4.68
0.89
0.12
na
0.04
2.49
0.63
0.36
0.12
4.94
1.31
0.13
na
0.09
2.91
1.02
0.70
0.10
4.03
1.16
0.13
na
9a
9b
9c
9d
9e
na
na
4.99
na
na
na
na
na
na
na
na
4.46
na
na
The resemblance among 1, its analogues 2, 3c, and known
telomerase inhibitors, together with the fact that most of the
human tumors seem to depend on telomerase reactivation to
prevent critical telomere loss, is testament to the potential of
exploring these compounds as anticancer therapies.
na
a na: not active at 10 µg/mL.
Compared to the control cells (Figure 2, panels 1a-c), the
cells treated with 1 (Figure 2, panels 2a-c) and with compound
2 (Figure 2, panels 3a-c) had higher population stopped at the
G2 phase except for A-549 cells treated with compound 2 that
showed enlarged arrest in S phase. Nervertheless, cells treated
with compound 3c showed a very pronounced arrest in S phase
in all cell lines (Figure 2, panels 4a-c). Hence, these compounds
alter cell cycle progression.
Induction of Apoptosis by Thiopeptide 1, 2, or 3c in
HT-29, A-549, and MDA-MB-231 Cell Lines. Many antican-
cer drugs induce cell cycle arrest and apoptosis. Apoptotic cells
undergo characteristic morphological changes. Among these,
the cell surface often bends and breaks up into membrane-
enclosed fragments called apoptotic bodies. This process
depends on a cascade of proteolytic enzymes called caspases.15
They exist in most of the cells as inactive precursors (zymogens)
that, once activated, kill cells.
To analyze whether 1, 2, and 3c induce apoptosis, HT-29,
A-549, and MDA-MB-231 cell lines were incubated with each
compound and then qualitatively measured for apoptosis (see
Experimental Section). The analysis consisted of detecting active
caspases (caspase-3/7) using a colorimetric test (CaspaTag in
situ caspase detection kits, CHEMICON).
All cell lines showed a high level of apoptotic cell death after
treatment with 1 (Figure 3, panels 2a-c), compound 2 (Figure
3, panels 3a-c), or compound 3c (Figure 3, panels 4a-c). A
negative control of the assay was performed by analyzing
untreated cells under the same experimental conditions (Figure
3, panels 1a-c).
Experimental Section
For general data, see the Supporting Information.
Compounds 4a, 5a, 6, 7a, 8a, and 9a were synthesized as
previously reported.8
Macrocyclization-Elimination Reaction for Preparation of
1a-c. A solution of 9 (1.0 mmol) in 98% HCO2H (28 mL) was
stirred under reflux for 1 h. The reaction mixture was cooled, poured
into a solution of NaHCO3, and then extracted with CH2Cl2. The
organic layer was dried and then concentrated in vacuo to give the
keto- and O-deprotected product as a yellow oil. A solution of this
oil in EtOH (70 mL) was stirred at 85 °C for 48 h. Concentration
in vacuo gave a brown residue, which was purified on the Isco
Flash system with a RediSep silica gel disposable flash column.
Elution with CH2Cl2-MeOH (9:1) gave the O-deprotected product
as a yellow oil. This product was dissolved in dry CH2Cl2 (20 mL),
and the solution was cooled to 0 °C. TEA (2.48 mmol) and MsCl
(1.24 mmol) were then added dropwise. The resulting solution was
stirred for 5 h at 0 °C, washed with NH4Cl and water, dried, and
concentrated.
Peptide-Heterocycle 1a. The final product was purified by
preparative HPLC using H2O (0.045% TFA) and MeCN (0.036%
TFA) as eluents (gradient 40-80% in 25 min; flow rate 3 mL/
min) to afford 1a (65.4 mg, 10%) as a yellow solid. Mp (CHCl3)
165-167 °C. [R]D +16.8 (c 0.25, DMF). 1H NMR (DMF, 400
MHz) δ 0.81-1.07 (m, 12H); 1.22-1.32 (m, 1H); 1.52-1.69 (m,
1H); 1.92-2.02 (m, 1H); 2.04-2.15 (m, 1H); 2.77 (s, 3H); 4.42
(dd, J ) 6.8 and 9.6 Hz, 1H); 4.79-4.82 (m, 1H); 5.69 (s, 1H);
6.19 (s, 1H); 7.50-7.61 (m, 3H); 8.50-8.53 (m, 2H); 8.59 (s, 1H);
8.64 (bs, 1H); 8.91 (bs, 1H); 9.16 (s, 1H); 9.19 (s, 1H); 10.22 (bs,
1H). 13C NMR (DMF, 100 MHz) δ 11.4 (q); 11.7 (q); 13.6 (q);
18.7 (q); 19.3 (q); 26.7 (t); 31.8 (d); 39.7 (d); 55.6 (d); 60.6 (d);
104.5 (t); 121.3 (d); 127.0 (s); 127.9 (2d); 128.0 (s); 128.2 (s);
128.6 (2d); 129.3 (s); 130.2 (d); 130.7 (s); 136.0 (s); 138.1 (s);
139.5 (d); 140.4 (d); 143.0 (s); 151.5 (s); 153.6 (s); 155.5 (s); 155.8
(s); 156.5 (s); 157.5 (s); 157.9 (s); 171.0 (s); 172.2 (s). MS (MALDI-
TOF) m/z 761 (M + K, 100), 745 (M + Na, 85). HRMS m/z calcd
for C36H38N9O7S (M + NH4) 740.2609, found 740.2602.
Conclusion
Several analogues of thiopeptide 1 were synthesized using
slight variations of a methodology previously described for the
preparation of the parent compound. Conditions that allow
isolation of the hydroxymethyl derivative have been established,
thus enabling preparation of several analogues by acylation.
Methyl substituents were introduced onto the pentaazole system
by employing different starting materials, whereas the exocyclic
double bond was modified in the last synthetic step.
Thiopeptide 1 and analogues 2 and 3c were shown to inhibit
cell cycle progression in cancer cell lines. As previously
mentioned, 1 is closely related in structure to YM-2163919 and
the potent telomerase inhibitor telomestatin.5
Peptide-Heterocycle 1b. The final product was purified by
preparative HPLC using H2O (0.045% TFA) and MeCN (0.036%
TFA) as eluents (gradient 40-80 in 25 min; flow 3 mL/min) to
afford 1b (51.1 mg, 7%) as a yellow solid. Mp (CHCl3) 160-162
°C. [R]D +18.4 (c 0.19, DMF). 1H NMR (DMF, 400 MHz) δ
0.81-1.01 (m, 12H); 1.21-1.33 (m, 1H); 1.48-1.61 (m, 1H);
1.95-2.03 (m, 1H); 2.06-2.16 (m, 1H); 2.94 (s, 3H); 4.41 (dd, J
) 6.4 and 9.4 Hz, 1H); 4.81-4.84 (m, 1H); 5.77 (s, 1H); 6.22 (s,
1H); 7.49-7.62 (m, 3H); 8.51 (s, 1H); 8.54-8.56 (m, 2H); 8.64
(bs, 1H); 8.90 (bs, 1H); 9.0 (s, 1H); 9.14 (s, 1H); 10.30 (bs, 1H).
13C NMR (DMF, 100 MHz) δ 11.2 (q); 11.7 (q); 13.6 (q); 18.7
Telomerase activity is important during telomere replication
in chromosomes; thus, telomerase inhibition will cause replica-
tion faults and DNA damage effect, which would in turn affect

5726 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Hernández et al.
Figure 2. Cell cycle progression in cancer cell lines treated with 1, 2 or 3c; a-1, HT-29 asynchronous colon cell line; a-2, HT-29 with thiopeptide
1; a-3, HT-29 with compound 2; a-4, HT-29 with compound 3c; b-1, A-549 asynchronous lung cell line; b-2, A-549 with thiopeptide 1; b-3, A-549
with compound 2; b-4, A-549 with compound 3c; c-1, MDA-MB-231 asynchronous breast cell line; c-2, MDA-MB-231 with thiopeptide 1; c-3,
MDA-MB-231 with compound 2; c-4, MDA-MB-231 with compound 3c.
(q); 19.3 (q); 26.7 (t); 31.7 (d); 39.8 (d); 55.6 (d); 60.7 (d); 105.4
(t); 125.6 (d); 127.2 (s); 127.8 (2d); 128.1 (s); 128.5 (s); 128.7
(2d); 129.3 (s); 130.1 (d); 132.4 (s); 135.4 (s); 136.5 (s); 139.5 (d);
140.7 (d); 143.6 (s); 151.4 (s); 153.3 (s); 156.2 (s); 158.4 (s); 159.0
(s); 159.6 (s); 160.3 (s); 171.1 (s); 172.3 (s). MS (MALDI-TOF)
m/z 761 (M + K, 100), 745 (M + Na, 85). HRMS m/z calcd for
C36H38N9O7S (M + NH4) 740.2609, found 740.2585.
Peptide-Heterocycle 1c. The final product was purified by
preparative HPLC using H2O (0.045% TFA) and MeCN (0.036%
TFA) as eluents (gradient 50-90% in 25 min; flow rate 3 mL/
min) to afford 1c (75 mg, 8%) as a yellow solid. Mp (CHCl3)
155-157 °C. [R]D +18.6 (c 0.16, DMF). 1H NMR (DMF, 500
MHz) δ 0.81-1.08 (m, 12H); 1.20-1.33 (m, 1H); 1.47-1.59 (m,
1H); 1.95-2.02 (m, 1H); 2.08-2.16 (m, 1H); 2.79 (s, 3H); 2.95
(s, 3H); 4.42 (dd, J ) 5.6 and 11.4 Hz, 1H); 4.82 (m, 1H); 5.70 (s,
1H); 6.19 (s, 1H); 7.53-7.61 (m, 3H); 8.51 (s, 1H); 8.56-8.57
(m, 2H); 8.63 (bs, 1H); 8.93 (bs, 1H); 9.16 (s, 1H); 10.26 (bs, 1H).
13C NMR (DMF, 125 MHz) δ 11.6 (q); 11.9 (q); 12.1 (q); 14.3
(q); 14.5 (q); 19.8 (q); 28.9 (t); 31.3 (d); 38.6 (d); 58.0 (d); 61.0
(d); 103.3 (t); 124.9 (d); 128.7 (2d); 129.6 (2d); 130.8 (d); 138.6
(d). MS (MALDI-TOF) m/z 775 (M + K, 100), 759 (M + Na,
85). HRMS m/z calcd for C37H40N9O7S (M + NH4) 754.2765, found
754.2764.

Cyclic Thiopeptide Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5727
Figure 3. Analysis of apoptotic cell death in cancer cells treated with thiopeptide 1, 2, or 3c: a-1, HT-29 colon cell line negative control; a-2,
HT-29 with compound thiopeptide 1; a-3, HT-29 with compound 2; a-4, HT-29 with compound 3c; b-1, A-549 lung cell line negative control; b-2,
A-549 with compound thiopeptide 1; b-3, A-549 with compound 2; b-4, A-549 with compound 3c; c-1, MDA-MB-231 breast cell line negative
control; c-2, MDA-MB-231 with compound thiopeptide 1; c-3, MDA-MB-231 with compound 2; c-4, MDA-MB-231 with compound 3c.
Peptide-Heterocycle 2. A solution of 9e (0.85 g, 1.01 mmol) in
98% HCO2H (25 mL) was refluxed for 1 h. The cooled reaction
mixture was poured into a solution of NaHCO3 and extracted with
CH2Cl2. The organic layer was dried and concentrated in vacuo to
give the keto-deprotected product as a yellow oil. A solution of
the oil in ethanol (80 mL) was stirred at 85 °C for 48 h.
Concentration in vacuo gave a brown residue, which was purified
by chromatography on silica gel. Elution with CH2Cl2-MeOH (100
to 95:5) and final purification were achieved by Isco Flash (4.3 g
reverse phase C18 RediSep column), H2O (0.04% TFA) and MeCN
(0.04% TFA) as eluents (gradient 5-20% in 40 min; flow rate 13
mL/min), to afford 2 (73 mg, 11%) as a yellow solid. Mp (CHCl3)
185-187 °C. [R]D +40.1 (c 3.13, DMSO). 1H NMR (DMSO, 400
MHz) δ 0.85-0.92 (m, 9H); 0.98 (d, J ) 6.8 Hz, 3H); 1.03-1.10
(m, 2H); 1.58-1.64 (m, 1H); 2.02-2.15 (m, 1H); 4.19-4.24 (m,
1H); 4.44-4.50 (m, 1H); 4.69-4.74 (m, 1H); 4.98 (dd, J ) 9.2
and 16.8 Hz, 1H); 7.51-7.58 (m, 4H); 8.33 (d, J ) 7.2 Hz, 2H);
8.40 (d, J ) 7.2 Hz, 1H); 8.52 (s, 1H); 8.67 (d, J ) 8.8 Hz, 1H);
8.8 (s, 1H); 9.05 (s, 1H); 9.12 (s, 1H). 13C NMR (DMSO, 100
MHz) δ 11.8 (q); 14.5 (q); 17.5 (q); 19.6 (q); 25.4 (t); 31.1 (d);
55.2 (d); 57.0 (d); 57.5 (d); 69.5 (t); 121.2 (d); 126.4 (s); 127.2
(d); 127.5 (s); 128.4 (2d); 129.1 (s); 129.4 (s); 129.9 (2d); 135.4
(s); 139.0 (d); 139.5 (d); 140.1 (s); 141.2 (d); 150.5 (s); 152.0 (s);
154.9 (s); 157.3 (s); 159.8 (s); 162.6 (s); 170.0 (s); 170.2 (s); 170.6
(s). MS (ES) m/z 714 (M + NH4, 100), 316 (5), 288 (18). HRMS
m/z calcd for C34H33N8O7S 697.2187 (M + H), found 697.2187.
Macrocyclization-Acylation for Preparation of 3a-d. A
solution of 9 (0.72 mmol) in 98% HCO2H (20 mL) was stirred
under reflux for 1 h. The reaction mixture was cooled, poured into
a solution of NaHCO3, and then extracted with CH2Cl2. The organic
layer was dried, and concentrated to give the keto- and O-
deprotected product as a yellow oil. A solution of the oil in EtOH
(40 mL) was stirred at 85 °C for 48 h. Concentration in vacuo
gave a brown residue, which upon elution with CH2Cl2-MeOH
(9:1) gave the alcohol 3 as a yellow oil.
Peptide-Heterocycle 3a. The alcohol 3 (105 mg, 0.14 mmol)
was dissolved in dry THF (20 mL). Ac2O (0.45 mL, 4.82 mmol)
was then added dropwise. The resulting solution was stirred for
8 h at room temperature, washed with NaHCO3, dried, and con-
centrated. The final product was purified by preparative HPLC using
H2O (0.04% TFA) and MeCN (0.04% TFA) as eluents (gradient
20-40% in 50 min; flow rate 15 mL/min) to afford 3a (15 mg,
13%) as a yellow solid. Mp (CHCl3) 171-173 °C. [R]D +71.8 (c
0.47, CHCl3). 1H NMR (CDCl3, 400 MHz) δ 0.93-1.01 (m, 12H);
1.28-1.35 (m, 2H); 1.63-1.75 (m, 1H); 1.85-1.95 (m, 1H); 2.04
(s, 3H); 3.59-3.68 (m, 1H); 4.32-4.49 (m, 1H); 4.65-4.75 (m,
2H); 5.75-5.84 (m, 1H); 6.15-6.24 (bs, 1H); 7.42-7.53 (m, 4H);
7.95 (s, 1H); 8.19 (s, 1H); 8.26 (s, 2H); 8.36 (d, J ) 7.2 Hz, 2H);
8.55 (bs, 1H). 13C NMR (CDCl3, 100 MHz) δ 11.8 (q); 14.5 (q);
18.8 (q); 18.9 (q); 20.4 (q); 25.7 (d); 29.6 (t); 30.1 (d); 39.7 (d);
47.0 (d); 56.5 (d); 63.6 (t); 119.6 (d); 126.5 (s); 127.2 (s); 128.0
(d); 128.5 (2d); 129.4 (s); 130.0 (s); 130.2 (s); 130.3 (2d); 135.7
(d); 137.4 (s); 137.7 (d); 139.8 (d); 141.8 (s); 151.4 (s); 153.0 (s);
156.0 (s); 158.2 (s); 160.4 (s); 162.4 (s); 170.8 (s); 171.7 (s); 172.0
(s). MS (ES) m/z 786 (M + NH4, 100), 744 (12), 316 (12), 288
(38), 241 (12). HRMS m/z calcd for C37H37N8O9S 769.2399 (M +
H), found 769.2409.
Peptide-Heterocycle 3b. The alcohol 3 (87 mg, 0.12 mmol) was
dissolved in dry CH2Cl2 (10 mL). 2-(2-Methoxyethoxy)acetic acid
(0.156 mL, 1.37 mmol), EDC ·HCl (263 mg, 1.37 mmol), and
DMAP (25 mg, 0.21 mmol) were then added. The resulting solution
was stirred for 8 h at room temperature, washed with NaHCO3,

5728 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Hernández et al.
dried, and concentrated. The final product was purified by prepara-
tive HPLC using H2O (0.04% TFA) and MeCN (0.04% TFA) as
eluents (gradient 20-38% in 70 min; flow rate 15 mL/min) to afford
3b (13 mg, 13%) as a yellow solid. Mp (CHCl3) 145-147 °C. [R]D
-30.2 (c 0.61, CHCl3). 1H NMR (CDCl3, 400 MHz) δ 0.84-1.07
(m, 12H); 1.26-1.35 (m, 2H); 1.52-1.75 (m, 1H); 1.81-1.96 (m,
1H); 3.34 (s, 3H); 3.53-3.71 (m, 6H); 4.09-4.22 (m, 2H);
4.46-4.56 (m, 1H); 4.59-4.81 (m, 1H); 5.55 (bs, 1H); 5.73-5.80
(m, 1H); 7.41-7.51 (m, 4H); 7.94 (s, 1H); 8.20 (s, 1H); 8.24 (s,
1H); 8.27 (s, 1H); 8.34 (d, J ) 8.0 Hz, 2H); 8.57 (bs, 1H). 13C
NMR (CDCl3, 100 MHz) δ 11.8 (q); 14.2 (q); 18.7 (q); 18.8 (q);
25.8 (d); 29.6 (t); 39.6 (d); 46.7 (d); 56.3 (d); 58.8 (q); 59.7 (d);
67.9 (t); 69.9 (t); 70.5 (t); 71.6 (t); 119.6 (d); 126.5 (s); 127.9 (s);
128.4 (d); 128.5 (2d); 129.9 (s); 130.3 (s); 130.3 (s); 130.4 (2d);
135.7 (s); 137.5 (d); 137.7 (d); 139.7 (d); 139.9 (s); 141.8 (s); 151.3
(s); 152.9 (s); 158.0 (s); 158.3 (s); 160.4 (s); 170.3 (s); 171.8 (s);
172.0 (s). MS (ES) m/z 860 (M + NH4, 100), 744 (55), 726 (45),
316 (45), 288 (98), 242 (22). HRMS m/z calcd for C40H43N8O11S
843.2750 (M + H), found 843.6727.
Peptide-Heterocycle 3c. The alcohol 3 (111 mg, 0.15 mmol)
was dissolved in dry CH2Cl2 (10 mL). 2-[2-(2-Methoxyethoxy)-
ethoxy]acetic acid (0.25 mL, 1.65 mmol), EDC ·HCl (316 mg, 1.65
mmol), and DMAP (30 mg, 0.25 mmol) were then added dropwise.
The resulting solution was stirred for 8 h at room temperature,
washed with NaHCO3, dried, and concentrated. The final product
was purified by preparative HPLC using H2O (0.04% TFA) and
MeCN (0.04% TFA) as eluents (gradient 20-38% in 70 min; flow
rate 15 mL/min) to afford 3c (16 mg, 12%) as a yellow solid. Mp
(CHCl3) 139-141 °C. [R]D -63.4 (c 0.56, CHCl3). 1H NMR (DMF,
400 MHz) δ 0.93-0.98 (m, 6H); 1.01 (d, J ) 6.8 Hz, 3H); 1.07
(d, J ) 6.8 Hz, 3H); 1.24-1.30 (m, 2H); 1.38 (d, J ) 6.4 Hz, 1H);
1.54-1.74 (m, 1H); 2.11-2.28 (m, 1H); 3.28 (s, 3H); 3.55-3.70
(m, 10H); 4.08-4.20 (m, 2H); 4.70-4.79 (m, 2H); 5.82 (s, 1H);
6.17 (s, 1H); 7.49-7.61 (m, 3H); 8.44 (d, J ) 8.0 Hz, 2H); 8.59
(s, 1H); 8.98 (s, 1H); 9.14 (s, 1H); 9.17 (s, 1H); 10.03 (bs, 1H).
13C NMR (DMF, 100 MHz) δ 11.8 (q); 14.5 (q); 18.9 (q); 19.2
(q); 26.8 (d); 29.4 (t); 32.4 (d); 39.4 (d); 58.1 (d); 58.2 (q); 60.1
(d); 68.1 (t); 68.3 (t); 70.3 (t); 70.4 (t); 70.5 (t); 71.9 (t); 121.6 (d);
127.3 (s); 127.9 (2d); 128.4 (s); 128.7 (s); 128.8 (2d); 129.4 (s);
130.3 (s); 130.4 (d); 136.5 (s); 139.8 (d); 140.7 (d); 140.8 (d); 142.2
(s); 152.8 (s); 155.8 (s); 158.1 (s); 158.5 (s); 159.6 (s); 160.7 (s);
171.0 (s); 171.6 (s); 171.9 (s). MS (ES) m/z 904 (M + NH4, 40),
726 (100), 316 (8), 288 (38). HRMS m/z calcd for C42H47N8O12S
887.3029 (M + H), found 887.3037.
Peptide-Heterocycle 3d. The alcohol 3 (50 mg, 0.068 mmol)
was dissolved in dry CH2Cl2 (7 mL). Boc-L-Arg-(Alloc)2-OH (120
mg, 0.27 mmol), EDC ·HCl (52 mg, 0.27 mmol), and DMAP (7
mg, 0.57 mmol) were added dropwise. The resulting solution was
stirred for 31 h at room temperature, washed with NaHCO3 and
NH4Cl, dried, and concentrated. The final product was purified by
flash chromatography on silica Bondesil C8 using H2O (0.04%
TFA) and MeCN (0.04% TFA) as eluents (gradient 0-50%) to
afford 3d (15.9 mg, 16%) as a yellow solid. [R]D -11.6 (c 0.82,
DMSO). 1H NMR (DMSO, 400 MHz) δ 0.82-0.94 (m, 9H); 0.99
(d, J ) 6.8 Hz, 3H); 1.07-1.17 (m, 2H); 1.25 (s, 9H); 1.35-1.37
(m, 2H); 1.57-1.64 (m, 2H); 1.73-1.78 (m, 1H); 2.01-2.11 (m,
1H); 3.09-3.16 (m, 2H); 3.57-3.65 (m, 1H); 3.79-3.91 (m, 3H);
4.47-4.51 (m, 2H); 4.62-4.70 (m, 3H); 4.75-4.79 (m, 1H);
5.12-5.40 (m, 4H); 5.87-5.99 (m, 2H); 7.08 (bs, 1H); 7.17 (bs,
1H); 7.30 (bs, 1H); 7.48-7.59 (m, 3H); 8.30-8.33 (m, 2H); 8.51
(s, 1H); 8.53 (s, 1H); 8.79 (bs, 1H); 8.98 (s, 1H); 9.11 (s, 1H);
9.12 (s, 1H); 9.98 (bs, 1H). 13C NMR (DMSO, 100 MHz) δ 12.3
(q); 14.2 (q); 17.9 (q); 18.5 (q); 19.0 (q); 22.4 (d); 26.9 (t); 28.1
(t); 28.9 (t); 31.9 (d); 41.7 (t); 53.4 (2d); 56.9 (d); 58.1 (d); 65.1
(t); 67.0 (t); 69.7 (t); 78.1 (s); 117.1 (s); 117.2 (t); 118.3 (t); 121.2
(d); 126.4 (s); 127.2 (s); 127.5 (s); 127.8 (s); 128.6 (2d); 129.1 (s);
129.6 (2d); 129.7 (s); 130.7 (d); 131.8 (d); 133.5 (s); 133.6 (d);
135.6 (s); 139.5 (d); 140.4 (d); 141.4 (d); 150.4 (s); 152.1 (s); 154.7
(s); 157.4 (s); 157.5 (s); 158.9 (s); 160.0 (s); 170.2 (s); 170.7 (s);
170.9 (s); 173.8 (s). MS (MALDI-TOF) m/z 1189 (M + K), 1173
(M + Na, 15), 1151 (M, 10). HRMS m/z calcd for C54H63N12O15S
1151.4295 (M + H), found 1151.4251.
Peptide-Heterocycle 3e. The O-deprotected product (74 mg,
0.101 mmol) was dissolved in dry CH2Cl2 (10 mL). Boc-L-Arg-
(Boc)2-OH (193 mg, 0.41 mmol), EDC ·HCl (78 mg, 0.41 mmol),
and DMAP (12.4 mg, 0.102 mmol) were then added dropwise. The
resulting solution was stirred for 15 h at room temperature, washed
with NaHCO3 and NH4Cl, dried, and concentrated. The final product
was purified by flash chromatography on silica Bondesil C8 using
H2O (0.04% TFA) and MeCN (0.04% TFA) as eluents (gradient
0-50%) to give 3e (19 mg, 17%) as a white solid. [R]D -19.4 (c
0.12, DMSO). 1H NMR (DMSO, 400 MHz) δ 0.83-0.95 (m, 12H);
1.09-1.18 (m, 2H); 1.23 (s, 9H); 1.36 (s, 9H); 1.43 (s, 9H);
1.45-1.52 (m, 4H); 1.60-1.68 (m, 1H); 1.96-2.03 (m, 1H);
3.95-4.04 (m, 1H); 4.11-4.18 (m, 2H); 4.39-4.48 (m, 2H);
4.63-4.69 (m, 1H); 5.63-5.70 (m, 1H); 7.14-7.28 (m, 2H);
7.50-7.75 (m, 3H); 8.17 (bs, 1H); 8.24 (bs, 1H); 8.39-8.41 (m,
2H); 8.55 (s, 1H); 8.72 (bs, 1H); 8.96 (s, 1H); 9.09 (s, 1H); 9.14
(s, 1H); 10.7 (bs, 1H). 13C NMR (DMSO, 100 MHz) δ 12.4 (q);
14.5 (q); 19.5 (q); 18.4 (q); 24.6 (d); 28.0 (q); 28.1 (t); 28.3 (t);
28.4 (q); 28.7 (q); 29.4 (t); 31.2 (d); 46.8 (d); 53.7 (d); 56.0 (d);
60.2 (t); 64.6 (t); 122.4 (d); 128.4 (2d); 129.3 (2d); 131.0 (d); 140.2
(d); 141.1 (d); 141.4 (d). MS (ES) m/z 1185 (M + 2H, 60), 1184
(M + H, 100). HRMS m/z calcd for C51H63N12O13S (M - Boc)
1083.4352, found 1083.4351.
Peptide-Heterocycle 3f. A solution of 3e (5 mg, 4.2 µmol) in
TFA-CH2Cl2 (25:75, 1 mL) was stirred at room temperature for
2 h. The TFA was removed, and the product was used without
1
purification. [R]D -40.1 (c 0.23, DMSO). H NMR (DMSO, 500
MHz) δ 0.83-0.98 (m, 12H); 1.07-1.18 (m, 2H); 1.41-1.76 (m,
5H); 2.03-2.08 (m, 1H); 3.57-3.64 (m, 1H); 4.0-4.04 (m, 1H);
4.08-4.18 (m, 3H); 4.49-4.56 (m, 2H); 4.65-4.68 (m, 1H);
5.69-5.73 (m, 1H); 7.51-7.62 (m, 7H); 8.20 (d, J ) 5.0 Hz, 1H);
8.39-8.41 (m, 2H); 8.56 (s, 1H); 8.71 (d, J ) 8.0 Hz, 1H); 8.80
(d, J ) 9.0 Hz, 1H); 8.99 (s, 1H); 9.10 (s, 1H); 9.14 (s, 1H). 13C
NMR (DMSO, 125 MHz) δ 12.5 (q); 14.4 (q); 19.5 (q); 19.7 (q);
25.0 (t); 25.1 (t); 26.0 (t); 26.8 (d); 30.4 (d); 46.9 (d); 56.2 (d);
58.4 (d); 60.3 (t); 65.2 (t); 70.4 (d); 122.3 (d); 128.4 (2d); 129.3
(2d); 131.0 (d); 139.6 (d); 140.9 (d); 141.0 (d). MS (ES) m/z 883
(M, 100). HRMS m/z calcd for C41H47N12O9S 883.3304, found
883.3317.
Peptide-Heterocycle 9b. Reaction (20 h) of the free acid 7a
(816 mg, 1.28 mmol) and 8b (500 mg, 1.54 mmol) using the general
procedure for amide formation gave 9b (1.14 g, 94%) as a solid.
An analytical sample was purified by column chromatography (4:1
to 2:3 hexane-EtOAc) to afford 9b as a yellow solid. Mp (CHCl3)
1
143-145 °C. [R]D +1.4 (c 0.42, CHCl3). H NMR (CDCl3, 400
MHz) δ 0.91-1.02 (m, 12H); 1.07 (s, 9H); 1.19-1.29 (m, 1H);
1.48-1.58 (m, 1H); 2.07-2.15 (m, 1H); 2.20-2.27 (m, 1H); 2.58
(s, 3H); 3.37 (s, 6H); 3.67-3.73 (m, 1H); 3.76-3.80 (m, 1H); 3.89
(s, 2H); 4.46-4.50 (m, 1H); 4.58-4.63 (m, 1H); 5.24-5.37 (m,
1H); 6.70 (d, J ) 8.4 Hz, 1H); 7.17 (bs, 1H); 7.38-7.46 (m, 4H);
7.80 (d, J ) 8.4 Hz, 1H); 8.24-8.30 (m, 2H); 8.36 (s, 1H); 8.37
(s, 1H); 8.47 (bs, 1H). 13C NMR (CDCl3, 100 MHz) δ 11.6 (q);
11.7 (q); 14.8 (q); 17.7 (q); 19.3 (q); 26.3 (t); 27.2 (q); 30.9 (d);
31.7 (t); 37.2 (d); 48.4 (d); 50.2 (2q); 57.4 (d); 58.3 (d); 62.2 (t);
73.9 (s); 99.3 (s); 124.3 (s); 126.5 (s); 128.3 (2d); 128.5 (2d); 129.6
(s); 130.2 (d); 140.0 (d); 141.2 (s); 142.6 (s); 143.0 (d); 151.3 (s);
151.7 (s); 153.0 (s); 155.8 (s); 161.3 (s); 161.4 (s); 161.6 (s); 170.9
(s); 171.2 (s); 188.3 (s). MS (MALDI-TOF) 965 (M81Br + Na,
100), 963 (M79Br
+ Na, 85). HRMS m/z calcd for
C42H5479BrN8O10S 941.2861 and to C42H5681BrN8O10S 943.2850,
found 941.2867 and 943.2853.
Peptide-Heterocycle 9c. Reaction (20 h) of the free acid 7b (1
g, 1.54 mmol) and the N-deprotected 8a (573 mg, 1.84 mmol) using
the general procedure for amide formation gave 9c (1.38 g, 95%)
as a yellow solid. An analytical sample was purified by column
chromatography (1:1 to 2:3 hexane-EtOAc) to give 9c as a yellow
1
solid. Mp (CHCl3) 116-118 °C. [R]D +2.3 (c 0.61, CHCl3). H
NMR (CDCl3, 400 MHz) δ 0.88-0.98 (m, 9H); 1.01 (d, J ) 6.8
Hz, 3H); 1.06 (s, 9H); 1.18-1.31 (m, 1H); 1.46-1.61 (m, 1H);

Cyclic Thiopeptide Analogues
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5729
2.05-2.16 (m, 1H); 2.19-2.27 (m, 1H); 2.78 (s, 3H); 3.36 (s, 6H);
3.67-3.75 (m, 1H); 3.77-3.81 (m, 1H); 3.87 (s, 2H); 4.45-4.66
(m, 2H); 5.32-5.42 (m, 1H); 6.79-6.88 (m, 1H); 7.36-7.46 (m,
3H); 7.78-7.89 (m, 2H); 8.07 (s, 1H); 8.12-8.17 (m, 1H);
8.24-8.29 (m, 2H); 8.36 (s, 1H); 8.51 (bs, 1H). 13C NMR (CDCl3,
100 MHz) δ 11.6 (q); 12.1 (q); 14.7 (q); 17.7 (q); 19.3 (q); 26.4
(t); 27.2 (q); 30.8 (d); 31.8 (t); 37.2 (d); 48.5 (d); 50.2 (2q); 57.4
(d); 58.3 (d); 62.4 (t); 73.9 (s); 99.2 (s); 125.0 (s); 126.7 (s); 128.2
(2d); 128.3 (2d); 129.4 (s); 130.0 (d); 139.4 (d); 141.4 (s); 143.3
(d); 152.0 (s); 152.4 (s); 152.8 (s); 154.6 (s); 158.8 (s); 161.4 (s);
164.1 (s); 171.0 (s); 171.3 (s); 173.6 (s); 188.0 (s). MS (MALDI-
TOF) 965 (M81Br + Na, 100), 963 (M79Br + Na, 80). HRMS m/z
calcd for C42H5479BrN8O10S 941.2861, found 941.2870.
and used for Tz reference (see next paragraph). After that, cells
are treated with vehicle alone (control) or compounds at the
concentrations indicated. Treated cells are further incubated for 72 h,
and cytotoxic evaluation was performed by colorimetric analysis.
In brief, cells are washed twice with phosphate buffered saline
(PBS), fixed for 15 min in 1% glutaraldehyde solution, rinsed twice
in PBS, and stained in 0.4% SRB solution for 30 min at room
temperature. Cells are then rinsed several times in 1% acetic acid
solution and air-dried. SRB was then extracted in 10 mM trizma
base solution and the absorbance measured at 490 nm. Cell survival
is expressed as percentage of control cell growth.
Dose-response curves are obtained by using the NCI algorithm:
17 Tz ) number of control cells at time t0, C ) number of control
cells at time t, and T ) number of treated cells at time t.
If Tz < T < C (growth inhibition), then the result is 100 ×
([T - Tz]/[C - Tz]).
Peptide-Heterocycle 9d. Reaction (20 h) of the free acid 7b
(763 mg, 1.17 mmol) and 8b (458 mg, 1.41 mmol) using the general
procedure for amide formation gave 9d as a solid (1.05 g, 94%).
An analytical sample was purified by column chromatography (4:1
to 2:3 hexane-EtOAc) to give 9d as a yellow solid. Mp (CHCl3)
If T < Tz (net cell killing), then the result is 100 × ([T - Tz]/
Tz).
1
147-149 °C. [R]D +3.5 (c 0.76, CHCl3). H NMR (CDCl3, 400
After dose-curve generation, results are experessed as GI50, the
compound concentration that causes 50% cell growth inhibition,
compared to control cultures.
MHz) δ 0.91-1.03 (m, 12H); 1.09 (s, 9H); 1.22-1.31 (m, 1H);
1.47-1.58 (m, 1H); 2.08-2.17 (m, 1H); 2.21-2.30 (m, 1H); 2.58
(s, 3H); 2.79 (s, 3H); 3.37 (s, 6H); 3.68-3.73 (m, 1H); 3.77-3.80
(m, 1H); 3.88 (s, 2H); 4.43-4.54 (m, 1H); 4.57-4.63 (m, 1H);
5.24-5.37 (m, 1H); 6.69 (d, J ) 8.4 Hz, 1H); 7.10 (bs, 1H);
7.38-7.46 (m, 3H); 7.78-7.83 (m, 2H); 8.25-8.30 (m, 2H); 8.37
(s, 1H); 8.44 (bs, 1H). 13C NMR (CDCl3, 100 MHz) δ 11.6 (q);
11.7 (q); 12.1 (q); 14.7 (q); 17.7 (q); 19.3 (q); 26.4 (t); 27.3 (q);
30.9 (d); 31.8 (t); 37.1 (d); 48.4 (d); 50.3 (2q); 57.4 (d); 58.3 (d);
62.2 (t); 73.9 (s); 99.2 (s); 124.4 (s); 125.0 (s); 126.7 (s); 128.3
(2d); 128.4 (2d); 129.4 (s); 130.0 (d); 141.2 (s); 142.9 (d); 151.2
(s); 152.0 (s); 152.5 (s); 152.8 (s); 155.8 (s); 158.8 (s); 161.5 (s);
170.8 (s); 171.0 (s); 171.2 (s); 188.4 (s). MS (MALDI-TOF) 979
(M81Br + Na, 100), 977 (M79Br + Na, 85). HRMS m/z calcd for
C43H5679BrN8O10S 955.3018 and to C43H5681BrN8O10S 957.3007,
found 955.3028 and 957.3009.
Cell Cycle Analysis. Cell cycle analysis was performed by
propidium iodide staining to determine DNA content. Subconfluent
cells treated with 35 nM 1 or 172 nM 2 or 135 nM 3c for 12 h
were trypsinyzed, collected by centrifugation, resuspended in PBS,
and then fixed in 70% ethanol. The fixed cells were incubated with
5 mg/mL propidium iodide (Sigma), 0.1 mg/mL RNase A (Sigma),
and 0.1% Triton X-100 for 15 min at 37 °C and analyzed with a
Beckman Coulter Epics XL using the 488 nm line of argon laser.
The cell cycle profile was analyzed using Cell Quest software.
Apoptosis Assay. The methodology used is based on fluoro-
chrome inhibitors of caspases (FLICA; CaspaTag in situ caspase
detection kits, Chemicon International, U.S. and Canada). The
inhibitors are cell permeable and noncytotoxic. Once inside the cell,
the inhibitor covalently binds to the active caspase. This kit uses a
carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor
of caspase-3 (FAM-DEVD-FMK), which fluoresces green. When
added to a group of cells, the FAM-DEVD-FMK probe enters each
cell and covalently binds to a reactive cysteine residue in the large
subunit of the active caspase heterodimer, thereby inhibiting further
enzymatic activity.18
Peptide-Heterocycle 9e. Reaction (20 h) of the free acid 7a (1.37
g, 2.15 mmol) and 8c (580 mg, 2.59 mmol) using the general
procedure for amide formation gave 9e (1.78 g, 98%) as a solid.
An analytical sample was purified by column chromatography (1:1
to 2:3 hexane-EtOAc) to give 9e as a solid. Mp (CHCl3) 132-134
1
°C. [R]D +22.4 (c 0.49, CHCl3). H NMR (CDCl3, 400 MHz) δ
0.89-1.07 (m, 12H); 1.22-1.31 (m, 1H); 1.51-1.61 (m, 1H);
2.05-2.18 (m, 1H); 2.21-2.45 (m, 1H); 3.37 (s, 6H); 3.79-3.86
(m, 1H); 3.89 (s, 2H); 4.29-4.84 (m, 3H); 6.77-6.95 (m, 1H);
7.35-7.44 (m, 3H); 7.74-7.85 (m, 2H); 8.04 (s, 1H); 8.09-8.12
(m, 1H); 8.17-8.25 (m, 2H); 8.31 (s, 1H); 8.36 (s, 1H); 8.49 (bs,
1H). 13C NMR (CDCl3, 100 MHz) δ 11.3 (q); 15.0 (q); 17.5 (q);
19.5 (q); 26.0 (t); 29.8 (d); 31.6 (t); 36.4 (t); 36.6 (d); 50.3 (2q);
58.4 (d); 58.6 (d); 99.3 (s); 126.5 (s); 128.2 (2d); 128.4 (2d); 129.4
(s); 130.1 (s); 130.3 (d); 139.6 (d); 140.1 (d); 141.4 (s); 143.3 (d);
151.8 (s); 153.0 (s); 154.5 (s); 161.4 (s); 161.7 (s); 161.9 (s); 162.6
(s); 171.5 (s); 171.8 (s); 188.1 (s). MS (MALDI-TOF) 865 (M81Br
+ Na, 100), 863 (M79Br + Na, 85). HRMS m/z calcd for
C36H4279BrN8O9S 841.1973, found 841.1968.
Cell Lines and Culture. Human-derived established cell lines
used in this study were purchased from ATCC (American Type
Culture Collection): A-549, human lung carcinoma (ATCC no.
CCL-185), HT-29, human colorectal adenocarcinoma (ATCC no.
HTB-38), and MDA-MB 231, human breast adenocarcinoma
(ATCC no. HTB-26).
Subconfluent cells cultured on coverslips were treated with 35
nM 1 or 172 nM 2 or 135 nM 3c for 24 h. After treatment, 30X
FLICA reagent was added at 1:15 dilution in culture medium and
incubated for 1 h at 37 °C. The cells were washed with PBS and
analyzed using a Leica TCS SP2 laser scanning confocal spectral
microscope (Leica Microsystems Heidelberg GmbH, Mannheim,
Germany) with a band-pass filter excitation at 490 nm and emission
at 520 nm to view the green fluorescence of caspase-positive cells.
The green fluorescence signal was a direct measure of the amount
of active caspase-3/7 present in the cell at the time the reagent was
added.
Acknowledgment. This study was partially supported by
CICYT (Grant BQU 2006-03794), Generalitat de Catalunya,
and the Barcelona Science Park. We gratefully acknowledge
PharmaMar S.A. for performing the preliminary biological tests,
and the Networking Centre on Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN). We also thank Dr. L. M. Can˜edo
for her contributions to compound purification. D.H. thanks the
Ministerio de Educacio´n y Ciencia for a predoctoral fellowship.
All cell lines are maintained in DMEM (Dulbecco’s modified
Eagle’s medium) supplemented with 10% FBS (fetal bovine serum)
and 100 units/mL penicillin and streptomycin at 37 °C and 5%
CO2. Triplicate cultures were incubated for 72 h in the presence or
absence of test compounds (at 10 concentrations typically ranging
from 10 to 0.0026 µg/mL).
Supporting Information Available: Experimental details, char-
acterization of synthesized compounds, and a table of HPLC purities
of final compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
A colorimetric assay using sulforhodamine B (SRB) has been
adapted for a quantitative measurement of cell growth and viability,
following a previously described method.16 Cells are plated in 96-
well microtiter plates at a density of 5 × 103/well and incubated
for 24 h. One plate from each different cell line is fixed and stained
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