Synthesis and biological evaluation of dual action cyclo-RGD/SMAC mimetic conjugates targeting αvβ3/α vβ5 integrins and IAP proteins

Article abstract of DOI:10.1039/c4ob00207e

The rational design, synthesis and in vitro biological evaluation of dual action conjugates 11-13, containing a tumour targeting, integrin α_vβ₃/α_vβ₅ ligand portion and a pro-apoptotic SMAC mimetic portion (cyclo-RGD/SMAC mimetic conjugates) are reported. The binding strength of the two separate units is generally maintained by these dual action conjugates. In particular, the connection between the separate units (anchor points on each unit; nature, length and stability of the linker) influences the activity of each portion against its molecular targets (integrins α_vβ₃/ α_vβ₅ for cyclo-RGD, IAP proteins for SMAC mimetics). Each conjugate portion tolerates different substitutions while preserving the binding affinity for each target.

Full text of DOI:10.1039/c4ob00207e

Organic &
Biomolecular Chemistry
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Synthesis and biological evaluation of dual action
cyclo-RGD/SMAC mimetic conjugates targeting
αvβ3/αvβ5 integrins and IAP proteins†
Cite this: Org. Biomol. Chem., 2014,
12, 3288
M. Mingozzi,‡a L. Manzoni,‡b D. Arosio,b A. Dal Corso,a M. Manzotti,a F. Innamorati,a
L. Pignataro,a D. Lecis,c D. Delia,c P. Seneci*a and C. Gennari*a
The rational design, synthesis and in vitro biological evaluation of dual action conjugates 11–13, contain-
ing a tumour targeting, integrin αvβ3/αvβ5 ligand portion and a pro-apoptotic SMAC mimetic portion
(cyclo-RGD/SMAC mimetic conjugates) are reported. The binding strength of the two separate units is
generally maintained by these dual action conjugates. In particular, the connection between the separate
units (anchor points on each unit; nature, length and stability of the linker) influences the activity of each
portion against its molecular targets (integrins αvβ3/αvβ5 for cyclo-RGD, IAP proteins for SMAC mimetics).
Each conjugate portion tolerates different substitutions while preserving the binding affinity for each
target.
Received 26th January 2014,
Accepted 21st March 2014
DOI: 10.1039/c4ob00207e
www.rsc.org/obc
Cancer implies a huge variety of pathologically altered
mechanisms in the body, and cancer research works on hun-
Introduction
In the pharmaceutical industry, inhibition of a molecular dreds of putative molecular targets.7 Cancer cells either
target sometimes leads to a bypass of the target itself by the mutate the drug target or transport the drug outside the
diseased cells or organs (e.g. penicillins and resistance to beta- cancer cell (imatinib8 and taxol9 are known examples of resis-
lactamases1). Thus, there is an increasing need for multi- tance to marketed drugs). Cancer cells are less likely to develop
targeted therapies.2
resistance against a dual action compound simultaneously
Multi-targeting may be achieved by administering a cocktail directed against two essential molecular targets. We chose
of active ingredients, and cocktails active against HIV are an integrins/angiogenesis10 and inhibition of apoptosis proteins
example of clinical success.3 However, effective drug combi- (IAPs)/apoptosis11 because our research group has accumu-
nations may require different administration routes, or lated significant experience on both target classes in the past.
different residence times in the human body.4 Well tolerated
drugs may become harmful in combination with other active
principles, due to drug–drug interactions.5
Project rationale
Integrins αVβ3, αVβ5 and α5β1 play a key role in angiogenesis
and tumour development, and are accessible as cell surface
receptors interacting with extracellular ligands.12 They are
involved in angiogenesis, tumour progression and meta-
stasis.13 The tripeptide sequence Arg-Gly-Asp (RGD) is the
common motif used by endogenous ligands to recognize and
bind such integrins.
A dual action compound contains the chemical elements
required to interact with two molecular targets.6 A connection
is chosen for each pharmacophore unit, and a suitable spacer
separates the two units without disturbing their biological
activities.
Cyclo[Arg-Gly-Asp-Phe-N(Me)-Val] (cilengitide)14 (1, Fig. 1)
reached phase III clinical trials against glioblastoma multi-
forme. We reported cyclic RGD-based peptidomimetics,
either built on 4-substituted aza-bicyclo[4.3.0]nonanes (ABN)
(2, Fig. 1),15,16 or built on bifunctional diketopiperazines
(DKP) (3, Fig. 1),17 as potent inhibitors of the purified αVβ3
receptor.
aUniversità degli Studi di Milano, Dipartimento di Chimica, Via Golgi 19, I-20133
Milan, Italy. E-mail: pierfausto.seneci@unimi.it, cesare.gennari@unimi.it;
Fax: +39-02-50314072; Tel: +39-02-50314060, +39-02-50314091
bIstituto di Scienze e Tecnologie Molecolari, Consiglio Nazionale delle Ricerche,
Via Golgi 19, I-20133 Milano, Italy
cFondazione IRCCS Istituto Nazionale dei Tumori, Dipartimento di Oncologia
Sperimentale e Medicina Molecolare, Via Amadeo 42, I-20133 Milan, Italy
†Electronic supplementary information (ESI) available: Experimental protocols
Apoptosis is started by stimuli received from within the
cell, or from the external environment.18 Degradation of cellu-
lar protein components is carried out by initiator and execu-
for the synthesis of cyclic RGD ligand–SMAC mimetic conjugate 17 and 1H and
13C NMR spectra for all compounds. See DOI: 10.1039/c4ob00207e
‡These authors contributed equally to the project.
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Fig. 1 Cyclic RGD ligands of αVβ3 integrin {Cilengitide (1, 15-membered), cyclo[ABN-RGD] (2, 15-membered) and cyclo[DKP-RGD] (3, 17-mem-
bered)} and pro-apoptotic SMAC mimetics 4 (GDC-0152), 5–7.
tioner Cysteine ASPartic acid-specific proteASES (caspases).19 conjugates targeting both angiogenesis and apoptosis could
IAPs20 bind through their baculovirus inhibitor repeat (BIR) be created.
domains to the initiator CASP-9 (BIR3 domain, primary
Herein we present the synthesis and biological evaluation
binding site) and to the executioner CASP-3 and CASP-7 of a small series of cyclic RGD ligand–ABD SMAC mimetic
(linker-BIR2 domain, secondary site), block caspases in their dual action conjugates (Fig. 3). We explored (i) the influence of
inactive forms and antagonize apoptosis.21 Endogenous SMAC 4- (11, 12) and C-terminus (13) connections on the ABD SMAC
protein22,23 (Second Mitochondria-derived Activator of Cas- mimetic unit, (ii) the influence of the ring size of the cyclic
pases) binds to the BIR3/linker-BIR2 domains of IAPs24 and RGD ligand unit (15-membered compounds 11, 12, 17-mem-
prevents CASP-3/-7/-9 inactivation, restoring caspase-depen- bered compound 13), and (iii) the presence of an ester- (11) or
dent apoptosis.
of an amide-containing (12, 13) linker connecting the cyclic
Structural studies25,26 showed that the AVPI N-terminal RGD and ABD SMAC portions.
sequence of SMAC binds to IAPs with nanomolar affinity. Pro-
apoptotic AVPI mimetics such as 4 (Fig. 1) are undergoing
clinical evaluation as anticancer agents.27 We introduced
4-substituted aza-bicyclo[5.3.0]decane (ABD) derivatives (e.g.,
Results and discussion
Chemistry
5–7, Fig. 1), endowed with good cell-free potency against IAPs
and moderate cytotoxicity.28,29
Synthetic targets – rationale. Compounds 11 and 12 (Fig. 3)
Since αV integrins are overexpressed on the surface of
cancer cells, their ligands can be used as tumour-homing/anti-
angiogenic peptidomimetics for site-directed delivery of cyto-
toxic drugs.30 For example, the cyclic RGD ligand–doxorubicin
conjugate 8 31 (Fig. 2) is highly cytotoxic against cancer cells
overexpressing integrins αVβ3/αVβ5 on their membrane. We
reported good stability and in vivo efficacy in a mouse model
of human ovarian cancer for the cyclo[ABN-RGD]paclitaxel
conjugate 9 32 and the cyclo[DKP-RGD]paclitaxel conjugate
10 33 (Fig. 2).
are dual action conjugates where the cyclo[DKP-RGD] integrin
ligand unit 3 is coupled with the ABD SMAC mimetic unit 6.
The former unit bears a p-aminomethyl group on the DKP-
nitrogen benzyl substituent, while the latter unit is functiona-
lised either with a p-hydroxymethyl (11, X = O) or with a
p-aminomethyl group (12, X = NH) on the 4-benzylaminoethyl
substituent. Ideally, both substitutions on the cyclo[DKP-RGD]
ligand 333 and on the ABD SMAC mimetic 629 should not affect
their binding affinity to integrin αVβ3 and to IAPs, respectively.
1,4-Butanedioic acid is used as a relatively short linker,
because the distance between the DKP and the ABD scaffolds
(21 atoms, including two phenyl groups) is largely determined
We reasoned that, by connecting the AVPI/SMAC mimetic
unit (5–7) with a cyclic RGD ligand such as 2 or 3, dual action
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Fig. 2 Cyclic RGD ligand–doxorubicin conjugate 8, cyclo[ABN-RGD]paclitaxel 9 and cyclo[DKP-RGD]paclitaxel 10.
respective molecular targets.16,34 11-Aminoundecanoic acid
was used as a relatively long linker, to keep a suitable distance
(18 atoms) between the ABN and ABD scaffolds.
Cyclo[DKP-RGD]-ABD dual action compounds 11 and
12. Our synthetic strategy to the ester-connected conjugate 11
started from previously reported N-Boc protected amine 16
(Scheme 1).28 Its C-terminal CONH–CHPh2 amide contributes
to BIR/IAP binding,28,35 while its 4-aminoethyl function is
used as a connection for the linker, which was built as shown
in Scheme 1.
Namely, amine 1628 was submitted to a reductive alkylation
(steps b, c, Scheme 1) with 4-hydroxymethyl benzaldehyde 15
(obtained by selective reduction with sodium borohydride of
terephthalaldehyde 14, step a). The resulting aminoalcohol 17
was chemoselectively N-Boc protected (step d), and finally
bis-N-Boc protected benzyl alcohol 18 was acylated with succinic
anhydride (step e, Scheme 1) to yield the ester-connected con-
struct 19. The overall yield for the reaction steps from 16 to 19
was a rather good 62%. A single direct phase chromatographic
column (step c) was needed to purify the reaction products.
Construct 19 was then coupled to the previously reported
cyclo[DKP-RGD] derivative 2033 (steps a, b, Scheme 1), to give
the bis-N-Boc protected, ester-connected conjugate 21. The
multi-functional nature of both coupling partners and the
presence of potentially reactive functions (e.g., the guanidine
and carboxylate functions in 20) required the initial activation
of the carboxylic group of 19 (step f), followed by its coupling
Fig. 3 Heterodimeric cyclic RGD ligand-ABD SMAC mimetic dual
action conjugates: 4-connected cyclo[DKP-RGD]-ABD compounds
11–12 and C-terminus-connected cyclo[ABN-RGD]-ABD compound 13.
by the DKP-nitrogen p-aminomethylbenzyl and the ABD
4-(p-X-methylbenzylaminoethyl) substitutions.
Compound 13 (Fig. 3) was made by coupling a cyclo
[ABN-RGD] integrin ligand unit 2, where the original 4-hydro-
xymethyl substitution was replaced by 4-aminomethyl group, with the amino group of 20 (step g) under carefully controlled
with the ABD SMAC mimetic 5, where the pro-(S) phenyl of the
conditions. Namely, coupling at pH 7.5 largely prevented side
original C-terminal diphenylmethylamide was replaced by
reactions involving the unprotected functionalities of 20, and
a carboxylic group. Once again, ideally both substitutions provided an acceptable 40% overall yield. Finally, acidic hydro-
should be well tolerated in terms of binding affinities to the
lysis of the N-Boc groups led to quantitative deprotection and
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Scheme 1 Synthesis of cyclo[DKP-RGD]-ABD SMAC mimetic, ester-connected dual action compound 11. Reagents and conditions: (a) NaBH4,
EtOH–THF–H2O, −5° to 0 °C, 6 h, 83%; (b) 15, DIPEA, dry MeOH, rt, 5 h; (c) NaBH4, rt, 30 min, 80% (two steps); (d) Boc2O, DIPEA, dry CH2Cl2, 0 °C
to rt, 24 h, 87%; (e) succinic anhydride, DIPEA, DMAP, dry CH2Cl2, 0 °C to rt, 2 h, 90%; (f) 19, N-hydroxysulfosuccinimide, DIC, dry DMF, rt, 18 h;
(g) 20, CH3CN, phosphate buffer, pH 7.5, 0 °C, 18 h, 40% over two steps; (h) TFA, CH2Cl2, rt, 50 min, 99%.
isolation of target cyclo[DKP-RGD]-ABD, ester-connected dual tected by hydrogenolysis (step f), and finally bis-N-Boc pro-
action conjugate 11 (Scheme 1). HPLC purification was tected diamine 27 was acylated with succinic anhydride (step
required to obtain pure compounds 21 and 11.
g, Scheme 2) to yield the amide-connected construct 28. The
Our synthetic strategy to the amide-connected conjugate 12, overall yield for the reaction steps from 16 to 28 was an excel-
and in particular to the key bis-N-Boc protected carboxylate 28, lent 82%. A direct phase chromatographic column (step d) and
is shown in Scheme 2. Amine 1628 was submitted to a reduc- a reverse phase HPLC purification (step g) were required to
tive alkylation (steps c, d, Scheme 2) with (p-aminomethyl) obtain pure 28.
benzaldehyde 24, obtained in turn by chemoselective N-Cbz
Construct 28 was then coupled to the previously reported
protection of (p-aminomethyl)phenyl methanol 22 (step a), fol- cyclo[DKP-RGD] derivative 2033 (steps h, i, Scheme 2), to give
lowed by partial oxidation with manganese dioxide of (p-Cbz- the bis-N-Boc protected, amide-connected conjugate 29. Final
aminomethyl)phenyl methanol 23 (step b). The resulting acidic N-Boc deprotection (step j, Scheme 2) led to target cyclo
N-Cbz, N-Boc protected diamine 25 was N-Boc protected on its [DKP-RGD]-ABD, amide-connected dual action conjugate 12 in
free secondary amine (step e), its primary amine was depro- a good 49% overall yield.
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Scheme 2 Synthesis of cyclo[DKP-RGD]-ABD SMAC mimetic, amide-connected dual action compound 12. Reagents and conditions: (a) DIPEA,
Cbz-Cl, dry THF, 0 °C to rt, 18 h, 71%; (b) MnO2, dry THF, rt, 18 h, 97%; (c) 24, DIPEA, dry MeOH, rt, 5 h; (d) NaBH4, rt, 30 min, 83% over two steps;
(e) Boc2O, DIPEA, dry CH2Cl2, 0 °C to rt, 24 h, 100%; (f) H2, Pd/C 10%, THF–H2O, rt, 18 h, 100%; (g) succinic anhydride, DIPEA, DMAP, dry CH2Cl2,
0 °C to rt, 2 h, 99%; (h) N-hydroxysulfosuccinimide, DIC, dry DMF, rt, 18 h; (i) 20, CH3CN, phosphate buffer, pH 7.5, 0 °C, 18 h, 49% over two steps;
( j) TFA, CH2Cl2, rt, 50 min, 100%.
The experimental conditions and purification protocols for (Scheme 3). Its 4-(2-benzoylaminoethyl) substituent contrib-
each reaction step in Scheme 4 leading to conjugate 12 are utes to BIR/IAP binding.28 The C-terminal carboxylic acid is
identical to the corresponding steps described in Scheme 1. A the anchoring point for the linker.
comparison between the yields of corresponding steps (steps
The synthesis of N-Boc protected carboxylic acid 30 started
b–e, Scheme 1, and steps c–g, Scheme 2; steps a–c, Scheme 1 from the already reported tricyclic ester 31.28 Namely, simul-
and 2) shows better results for amide-connected conjugate 12. taneous hydrogenolytic isoxazolidine opening and benzyl
It is reasonable to hypothesize a lower stability of the ester deprotection (step a, Scheme 3) and chemoselective N-Boc pro-
bond in ester-connected conjugate 11, and in intermediates 19 tection (step b) provided N-protected alcohol 32. Mesylation
and 21, when compared with the amide bond in amide-con- (step c) and microwave-assisted nucleophilic substitution
nected conjugate 12, and in intermediates 28 and 29. This may (step d) led to N-protected aminonitrile 33. Acidic deprotection
lead to lower overall yields either under the reaction con- (step e, aminonitrile 34) was followed by amidation of the
ditions, or during the work up-purification protocols.
3-amino group with N-protected/methylated (S)-aminobutyric
trans-Cyclo[ABN-RGD]-ABD dual action compound 13. Our acid (step f), yielding nitrile 35. Nitrile reduction was carried
synthetic strategy to the amide-connected conjugate 13 out using the H-Cube™ continuous flow hydrogenation appar-
required the synthesis of N-Boc protected carboxylic acid 30 atus (step g), and the amino function of compound 36 was
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Scheme 3 Synthesis of the N-Boc protected carboxylic acid intermediate 30. Reagents and conditions: (a) HCOONH4, Pd(OH)2/C, THF–H2O 4/1,
2% AcOH, 70 °C, 6 h, then rt, 18 h; (b) Boc2O, TEA, dry CH2Cl2, rt, 20 h, 59% over two steps; (c) MsCl, TEA, dry CH2Cl2, 0 °C to rt, 2 h; (d) nBuN4CN,
dry DMF, MicroWave, 80 °C, 2 h, 84% over two steps; (e) AcCl, dry MeOH, rt, 5 min, 100%; (f) N-Boc, N-Me-(S)-2-aminobutyric acid (Abu), EDC·HCl,
HOBt, DIPEA, dry CH2Cl2, rt, 22 h, 82%; (g) H2, 60 bar, Ni-Ra cartridge, citric acid, EtOH–H2O, 60 °C, 5 h, continuous flow, 0.5 mL min−1, 24 h, 98%;
(h) BzCl, TEA, dry CH2Cl2, rt, 5 h, 92%; (i) LiOH, THF–H2O, 0 °C to rt, 5 h, 88%.
benzoylated (step h). Finally, methyl ester 37 was hydrolysed Biology
under basic conditions (step i, Scheme 3) to provide N-Boc pro-
tected carboxylic acid 30. The overall yield for the nine
Compound profiling – rationale. The cell-free potency of the
reaction steps from 31 to 30 was an acceptable 32% (average dual action conjugates 11–13 on the BIR domains of IAPs
reaction yield = 89%). Four direct phase chromatographic sep- (SMAC mimetic unit), and on integrins αvβ3 and αvβ5 (cyclic
arations (steps b, d, f, h) were needed to purify the reaction RGD ligand unit) was investigated to confirm the affinity for
products.
their molecular targets. Then, their cytotoxicity on tumour cell
The synthetic strategy designed to prepare the amide-con- lines characterized by various levels of cytoplasmic IAPs and
nected linker construct 38 is reported in Scheme 4. 11-Ami- membrane integrins was investigated.
noundecanoic acid 39 was esterified (step a, Scheme 4) and
Cell-free assays – BIR domains/IAPs. Three BIR domain-
the resulting aminoester 40 was coupled with N-Boc-(S)-phe- based IAP portions were used to test the affinity of the dual
nylglycine using classical peptide coupling conditions (step b). action conjugates 11–13. The primary BIR3 binding site/
Acidic deprotection (step c) of N-Boc protected 41 led to the domain from XIAP and cIAP2 was selected to measure the
aminoester linker 42 as a hydrochloride salt. The linker was affinity for two of the most therapeutically relevant IAPs.28,29,36
then coupled to N-Boc protected carboxylic acid 30 (step d) to The bi-functional linker-BIR2-BIR3 portion from XIAP was
provide ester 43. Hydrolysis of the methyl ester (step e) yielded selected to determine any SMAC dimer-like behaviour of the
the expected amide-connected construct 38. The ester 43 was dual action conjugates 11–13.37,38 The standard monomeric
also N-deprotected (step f, Scheme 4) to provide the standard SMAC mimetic amides 5 and 6 (Fig. 1), and esters 7 (Fig. 1)
compound 44 for biological purposes (vide infra, Table 1). The and 44 (Scheme 4) were tested for comparison. Compound 5
overall yield of the reaction steps involving ABD SMAC mimetic bears a 11, 12-like 4-benzylaminoethyl substituent, compound
intermediates (steps d, e; Scheme 4) was a moderate 37%. Two 6 bears a 13-like 4-benzoylaminoethyl substituent, compound
direct phase chromatographic separations (steps b, d) were 7 bears a C-terminus phenylglycinamide ester, while com-
required to purify the reaction products. The carboxylic acid pound 44 bears a C-terminus phenylglycinamido-linker ester
construct 38 was then coupled to previously reported cyclo substituent. Their IC50 values are reported in Table 1. The
[ABN-RGD]methylamine 4516 (step g, Scheme 4), to give the results clearly show that the attachment of a linker and a cyclic
N-Boc protected, amide-connected conjugate 46. Final acidic RGD ligand onto the SMAC mimetic unit does not significantly
N-Boc deprotection (step h, Scheme 4) led to target cyclo- affect its affinity for IAPs. Indeed, 4-connected dual action con-
[ABN-RGD]-ABD, amide-connected dual action conjugate 13 in jugates 11 and 12 and reference compounds 5 and 6 show
a low, unoptimized two-step 17% yield.
comparable affinity for mono- and bi-functional domains from
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Scheme 4 Synthesis of cyclo[ABN-RGD]-ABD SMAC mimetic, amide-connected dual action compound 13. Reagents and conditions: (a) SOCl2,
MeOH, rt to 80 °C, 4 h, 98%; (b) N-Boc-(S)-PheGly, EDC·HCl, HOBt, DIPEA, dry CH2Cl2, rt, 18 h, 72%; (c) 3 N HCl/dry MeOH, rt, 18 h, 97%; (d) 30,
EDC·HCl, HOBt, DIPEA, dry CH2Cl2, rt, 18 h, 63%; (e) LiOH, THF–H2O, 0 °C to rt, 5 h, 59%; (f) 3 N HCl/dry MeOH, rt, 4 h, quantitative; (g) HATU,
HOAt, DIPEA, dry DMF, rt, 10 min, then 45, rt, 72 h, 30%; (h): TFA–thioanisole–1,2-ethanedithiol–anisole 90 : 5 : 3 : 2, rt, 2 h, 57%.
XIAP. The same is true for C-terminus connected dual action
conjugate 13 and reference compounds 7 and 44. As expected,
cyclic RGD-based reference compounds 2 and 3 did not show
affinity for any BIR construct from IAPs.
Table 1 IC50s of compounds 2, 3, 5–7, 11–13, 44 on BIR portions from
IAPs
The higher affinity of dual action conjugates 11–13 for cIAP
proteins is consistent with what is generally observed for
monomeric SMAC mimetics.29
BIR3,
XIAP (nM)
l-BIR2-BIR3,
XIAP (nM)
BIR3,
cIAP2 (nM)
Compound
Cell-free assays – αvβ3/αvβ5 integrins. The dual action conju-
gates 11–13 were tested in vitro for their ability to inhibit bio-
tinylated vitronectin binding to the purified αvβ3 and αvβ5
receptors.32,33 The standard monomeric, integrin ligands 2
and 3 (Fig. 1), built respectively on a cyclo[ABN-RGD], 13-like
scaffold, and on a cyclo[DKP-RGD], 11, 12-like scaffold, were
tested for comparison. Their IC50 values are reported in
Table 2. The three dual action conjugates 11–13 show nano-
11
12
13
5
6
7
44
2
3
52.0 4.5
25.5 1.7
66.0 5.4
120.0 18.6
110.0 26.4
760.0 99.2
90.4 12.2
>10 000
50.5 7.8
57.5 5.8
175.8 27.5
55.0 13.1
27.0 12.4
190.0 49.1
109.8 27.1
>10 000
1.33 0.3
0.73 0.1
1.48 0.2
NT
NT
NT
1.57 0.2
NT
NT
>10 000
>10 000
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although no hydrolysis or SMAC unit release can easily take
place.
Table 2 Inhibition of biotinylated vitronectin binding to αvβ3 and αvβ5
receptors by compounds 2, 3, 5–7, 11–13, 44
As expected, reference cyclic RGD ligands 2 and 3 per se do
not show any cytotoxicity against integrin-rich IGROV-1
cells.32,33 The same is true for reference SMAC mimetics 5–7
and 44. Thus, we assumed that dual action conjugates 11–13
should also be inactive against IGROV-1 cells. Surprisingly,
C-terminus-amide-connected conjugate 13 shows cytotoxicity
on IGROV-1 cells, possibly suggesting an advantage for C-ter-
minus connected dual action compounds in terms of cellular
activity.
Compound
αvβ3 IC50 (nM)
αvβ5 IC50 (nM)
11
12
13
2
3
5
6
7
44
70.1 0.1
36.5 0.6
105.2 3.4
20.2 1.9
4.5 1.1
>10 000
>10 000
>10 000
>10 000
900 380
1500 700
649 25
205 34
149 25
>10 000
>10 000
>10 000
>10 000
molar affinity for both αvβ3 (stronger interaction) and αvβ5
integrin (weaker interaction), with a limited loss in binding
strength with respect to the reference compounds 2 and 3.
However, their residual affinity (nanomolar) should be
sufficient to target integrin-expressing tumour cells. As
expected, reference SMAC mimetics 5–7 and 44 did not show
affinity for the αvβ3 and αvβ5 receptors. Based on these promis-
ing cell-free results, we decided to examine the cytotoxic
activity of the three dual action conjugates 11–13 on two
different tumour cell lines.
Cytotoxicity – tumour cells. The dual action conjugates
11–13 were tested in cellular assays for their ability to kill
tumour cells, either expressing integrin receptors on their
surface or relying on IAP proteins to impair the physiological
apoptotic process. We selected ovarian IGROV-1 carcinoma
cells as representatives for the former, and breast cancer
MDA-MB-231 cells for the latter group. The standard mono-
meric, integrin ligands 2 and 3 (Fig. 1) and IAP inhibitors 5–7
and 44 (Fig. 1 and Scheme 4), were tested for comparison.
Their IC50 values are reported in Table 3. The results clearly
show that dual action conjugates 11–13 are endowed with
moderate cytotoxic activity. Namely, 4-ester-connected conju-
gate 11 and reference SMAC mimetic 5 are similarly potent
against MDA-MB-231 cells. The same is true for C-terminus-
connected conjugate 13 and reference SMAC mimetic 6. The
Conclusions
In this paper we reported the rational design, synthesis and
in vitro evaluation of cyclic RGD ligand–SMAC mimetic conju-
gates 11–13. We successfully confirmed their affinity for the
molecular target of each monomeric unit, and we ascertained
that the moderate cytotoxicity of their monomeric precursors
can be maintained. Some preliminary results (e.g., the cyto-
toxic activity for C-terminus-amide connected conjugate 13 on
IGROV-1 cells) indicate that the combination of a cyclic RGD
ligand-based tumour targeting/anti-angiogenic unit with a
SMAC mimetic-based pro-apoptotic unit may lead to synergi-
stic/enhanced anticancer effects.
More generally, we plan to investigate the influence of
linker composition (e.g., PEG-based or amides) and hindrance
(e.g., rigid/constrained linkers, short linkers), and of overall
physico-chemical (e.g., lipophilicity) and electronic properties
(e.g., 4-substituents on the ABD SMAC mimetic unit) on bio-
logical activity. We aim to establish a detailed SAR, in order to
evaluate the potential for cyclic RGD ligand–SMAC mimetic
conjugates as anticancer agents in vitro and in vivo.
Experimental
loss of cytotoxicity for 4-amide-connected conjugate 12 with Materials and methods
respect to 4-ester-connected conjugate 11 may be due to ester
Reactions requiring anhydrous conditions were carried out in
hydrolysis and SMAC mimetic unit release for the latter com-
pound, as observed by us with cyclic RGD ligand–paclitaxel
conjugates.33 Surprisingly, C-terminus-amide connected conju-
gate 13 shows moderate cytotoxicity on MDA-MB-231 cells,
flame-dried glassware, with magnetic stirring and under a
nitrogen atmosphere. Commercially available reagents were
used as received. Anhydrous solvents were purchased from
commercial sources and withdrawn from the container by
syringe, under a slight positive pressure of nitrogen. N-Boc,
N-Me-(S)-2-aminobutyric acid,28 SMAC amine 16,28 cyclo
[DKP-RGD]benzylamine 20,33 SMAC tricyclic ester 31,28 and
cyclo[ABN-RGD]methylamine 4516 were prepared according to
literature procedures. Their analytical data were in agreement
with those already published. Reactions were monitored by
analytical thin-layer chromatography (TLC) using silica gel 60
F254 pre-coated glass plates (0.25 mm thickness). Visualization
was accomplished by irradiation with a UV lamp and/or stain-
ing with a potassium permanganate alkaline solution or
ninhydrin. Flash column chromatography was performed
according to the method of Still and co-workers39 using Chromagel
Table 3 Cytotoxicity of compounds 11–13, 2–3, 5–7, 44
Compound
MDA-MB-231 (μM)
IGROV-1 (μM)
11
12
13
2
9.7 1.6
>25
20.5 2.2
>25
>25
>25
11.5 2.5
>25
>25
3
>25
5
6
7
13.5 1.6
8.1 0.9
>25
>25
>25
>25
44
>25
>25
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60 ACC (40–63 µm) silica gel. Proton NMR spectra were solution of NaHCO3 were added, then the mixture was
recorded on a spectrometer operating at 400.16 MHz. Proton extracted with AcOEt (7 mL, three times). The organic phase
chemical shifts are reported in ppm (δ) with the solvent refer- was dried over Na2SO4 and the solvent was evaporated at
ence relative to tetramethylsilane (TMS) employed as the reduced pressure. The crude product was purified by flash
internal standard. The following abbreviations are used to chromatography on silica gel (CH2Cl2–MeOH, 9 : 1 as eluent)
describe spin multiplicity: s = singlet, d = doublet, t = triplet, to afford the pure desired product 17 as a white foam (41 mg,
q = quartet, m = multiplet, br = broad signal, dd = doublet of 0.059 mmol, 80% yield).
1
doublets. Carbon NMR spectra were recorded on a spectro-
Rf = 0.3 (CH2Cl2–MeOH, 9 : 1); H NMR (400 MHz, THF-d8)
meter operating at 100.63 MHz, with complete proton decou- δ 8.27 (bs, 1H), 7.54 (bs, 1H), 7.40–7.00 (m, 14H), 6.21 (d, J =
pling. Carbon chemical shifts are reported in ppm (δ) relative 8.81 Hz, 1H) 4.62 (d, J = 6.53 Hz, 1H), 4.60–4.35 (m, 4H),
to TMS with the respective solvent resonance as the internal 4.00–3.70 (m, 4H), 2.84 (bs, 3H), 2.66 (bs, 1H), 2.52 (bs, 1H),
standard. HPLC purifications were performed on a Dionex 2.15 (m, 4H), 1.91 (m, 2H), 1.85–2.57 (m, 4H), 1.46 (s, 9H),
Ultimate 3000 instrument equipped with a Dionex RS Variable 1.47–1.31 (m, 4H), 0.87 (t, J = 7.32 Hz, 3H); 13C NMR
Wavelength Detector (column: Atlantis® Prep T3 OBD™ 5 μm (400 MHz, THF-d8) δ172.3, 171.5, 170.6, 157.0, 143.5, 143.3,
19 × 100 mm). High resolution mass spectra (HRMS) were 142.7, 129.2, 129.0, 128.9, 128.2, 128.1, 127.6, 127.5, 127.0,
performed on a Fourier Transform Ion Cyclotron Resonance 80.2, 80.1, 79.7, 67.9, 67.7, 67.4, 67.2, 66.9, 66.7, 66.6, 64.6,
(FT-ICR) Mass Spectrometer APEX II & Xmass software (Bruker 64.4, 53.4, 53.1, 49.3, 48.4, 47.1, 35.0, 34.9, 34.6, 34.4, 34.3,
Daltonics) – 4.7 T Magnet (Magnex) equipped with ESI source, 33.9, 33.7, 33.5, 31.8, 27.7, 27.4, 27.1, 25.5, 25.3, 25.1, 24.9,
available at CIGA (Centro Interdipartimentale Grandi Apparec- 24.7, 22.4, 22.2, 22.2; MS (ESI) m/z calcd for [C43H58N5O6]+:
chiature) c/o Università degli Studi di Milano. Low resolution 740.44 [M + H]+; found: 739.75.
mass spectra (MS) were recorded on a Waters Acquity™
UPLC-MS instrument (ESI source).
tert-Butyl ((S)-1-(((3S,6S,7R,9aS)-3-(benzhydrylcarbamoyl)-7-(2-
((tert-butoxycarbonyl)(4-(hydroxymethyl)benzyl)amino)ethyl)-5-
oxooctahydro-1H-pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxobutan-2-yl)-
(methyl)carbamate 18. A solution of compound 17 (41 mg,
0.056 mmol, 1 eq.) and DIPEA (19 µL, 0.112 mmol, 2 eq.) in
dry CH2Cl2 (0.6 mL) was stirred in an ice bath under nitrogen
atmosphere. A solution of Boc2O (17 mg, 0.079 mmol, 1.4 eq.)
in dry CH2Cl2 (0.2 mL) was added. The mixture was stirred at
0 °C for 1 h under nitrogen atmosphere, then stirring was con-
tinued overnight at room temperature. Then, additional Boc2O
(6.7 mg, 0.030 mmol, 0.5 eq.) was dissolved in CH2Cl2 (0.2 mL)
and added, and stirring continued for 4 h. The solvent was
removed at reduced pressure, and the crude product was
passed through a plug of silica gel (CH2Cl2–MeOH, 95 : 5 as
eluent) to afford the desired pure product 18 as a white foam
(41 mg, 0.049 mmol, 87% yield).
Chemistry
General procedure for the synthesis of compound 11
4-Hydroxymethylbenzaldehyde 15. Terephthalaldehyde 14
(1.0 g, 7.45 mmol, 1 eq.) was dissolved in 12.5 mL of EtOH–
H2O (95 : 5), and then 17.5 mL of THF were added. To the
resulting stirred solution, NaBH4 (70 mg, 1.86 mmol, 0.25 eq.)
was added at −5 °C in small portions over 30 minutes. The
mixture was stirred for 6 h, while the temperature was main-
tained at 0 °C. The reaction mixture was then adjusted to
pH = 5 with 2 M HCl. The solvent was evaporated at reduced
pressure, then H2O (20 mL) was added and the solution was
extracted three times with AcOEt. The resulting organic phase
was dried over Na2SO4 and the solvent was evaporated at
reduced pressure. The crude product was purified by flash
chromatography on silica gel (n-hexane–AcOEt, 1 : 1 as eluent)
to afford the pure desired product 15 as a white solid (840 mg,
6.18 mmol, 83%).
1
Rf = 0.7 (CH2Cl2–MeOH, 9 : 1); H NMR (400 MHz, acetone-
d6) δ 8.18 (d, J = 8.70 Hz, 1H), 7.40–7.08 (m, 14H), 6.19 (d, J =
8.66 Hz, 1H), 4.69 (d, J = 7.92 Hz, 1H), 4.61 (d, J = 5.76 Hz, 2H),
4.58–4.30 (m, 4H), 4.13 (t, J = 5.76 Hz, 1H), 3.99 (m, 1H), 3.25
(m, 1H), 2.25 (m, 1H), 2.17 (bs, 1H), 2.00–1.58 (m, 7H), 1.50
(s, 18H), 1.46 (m, 4H), 1.32 (m, 2H), 0.90 (t, J = 7.38 Hz, 3H);
MS (ESI) m/z calcd for [C48H66N5O8]+: 840.49 [M + H]+; found:
840.45.
Rf = 0.5 (Hex–AcOEt, 4 : 6); 1H NMR (400 MHz, CDCl3)
δ 9.82 (s, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.39 (d, J = 7.9 Hz, 2H),
4.65 (s, 2H), 2.07 (s, 1H); 13C NMR (101 MHz, CD2Cl2) δ 192.6,
148.8, 135.9, 130.2, 127.3, 64.5.
tert-Butyl
(2-((4
((S)-1-(((3S,6S,7R,9aS)-3-(benzhydrylcarbamoyl)-7-
(hydroxymethyl)benzyl)amino)ethyl)-5-oxooctahydro-1H- butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-
4-((4-(((2-((3S,6S,7R,9aS)-3-(Benzhydrylcarbamoyl)-6-((S)-2-((tert-
pyrrolo[1,2-a]azepin-6-yl)amino)-1-oxobutan-2-yl)(methyl)carbamate pyrrolo[1,2-a]azepin-7-yl)ethyl)(tert-butoxycarbonyl)amino)methyl)-
17. A solution of SMAC amine 1628 (50 mg, 0.074 mmol, benzyl)oxy)-4-oxobutanoic acid 19. Compound 18 (17 mg,
1.05 eq.), DIPEA (18 µL, 0.105 mmol, 1.5 eq.) in dry MeOH 0.020 mmol, 1 eq.) was dissolved in dry CH2Cl2 (0.12 mL).
(0.5 mL) was added to a flask containing compound 15 Then DIPEA (4.1 µL, 0.024 mmol, 1.2 eq.) was added and the
(9.5 mg, 0.074 mmol, 1 eq.) under nitrogen atmosphere. The mixture was stirred in an ice bath. DMAP (1.46 mg,
mixture was left stirring at room temperature for 5 h. NaBH4 0.012 mmol, 0.6 eq.) and succinic anhydride (3.6 mg,
(5.3 mg, 0.14 mmol,
2 eq.) was then added stepwise 0.036 mmol, 1.8 eq.) were added. The reaction was stirred at
(5 additions within 3 min), and the mixture was stirred at room temperature under nitrogen atmosphere for 2 h. CH2Cl2
room temperature under nitrogen for 30 minutes. The mixture (15 mL) was then added and the solution was washed with a
was then concentrated under vacuum. 5 mL of a saturated solution of 1 M KHSO4 (7 mL, twice). The aqueous phase was
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extracted with CH2Cl2 (7 mL, once), then the collected organic 43.9, 43.8, 42.2, 40.1, 40.1, 39.9, 37.8, 34.0, 33.6, 31.4, 31.1,
phases were dried over Na2SO4. The solvent was evaporated at 31.0, 30.8, 30.4, 28.9, 28.8, 27.7, 26.5, 23.0, 11.1; MS (ESI) m/z
reduced pressure to afford the desired product 19 as a white calcd for [C79H106N15O18]+: 1552.78 [M + H]+; found: 1552.85.
2-((1S,5S,11S,15R)-18-(4-((4-((4-(((2-((3S,6S,7R,9aS)-3-(Benzhy-
drylcarbamoyl)-6-((S)-2-(methylamino)butanamido)-5-oxoocta-
hydro-1H-pyrrolo[1,2-a]azepin-7-yl)ethyl)amino)methyl)benzyl)-
oxy)-4-oxobutanamido)methyl)benzyl)-11-(3-guanidinopropyl)-
4,7,10,13,17,19-hexaoxo-3,6,9,12,16,18-hexaazabicyclo[13.2.2]-
nonadecan-5-yl)acetic acid 11. Compound 21 (9 mg,
0.0058 mmol, 1 eq.) was dissolved in CH2Cl2 (100 μL). TFA
(66 μL, 0.87 mmol, 150 eq.) was then added, and stirring was
continued at room temperature for 50 minutes. The solvent
and TFA were removed at reduced pressure, and the crude
product was purified by HPLC (gradient: from 90% H2O +
0.1% CF3COOH–10% acetonitrile + 0.1% CF3COOH to 30%
H2O + 0.1% CF3COOH–70% acetonitrile + 0.1% CF3COOH in
14 min). The purified product was then freeze-dried to give the
trifluoroacetate salt of the desired pure compound 11 as a
white foam (9.7 mg, 0.0057 mmol, 99% yield).
foam that was used without further purification (17 mg,
0.018 mmol, 90% yield).
1
Rf = 0.6 (CH2Cl2–MeOH, 9 : 1); H NMR (400 MHz, acetone-
d6) δ 10.72 (bs, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.48–6.98 (m,
15H), 6.19 (d, J = 8.4 Hz, 1H), 5.12 (s, 2H), 4.69 (d, J = 7.1 Hz,
1H), 4.63–4.26 (m, 3H), 3.99 (d, J = 7.1 Hz, 1H), 3.27 (s, 1H),
3.03–2.93 (m, 2H), 2.80 (s, 3H), 2.70–2.54 (m, 2H), 2.36–2.21
(m, 1H), 2.16 (s, 1H), 2.02–1.57 (m, 6H), 1.49 (s, 18H),
1.45–1.35 (m, 6H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (400 MHz,
acetone-d6) δ 173.6, 172.7, 170.8, 143.6, 143.3, 139.7, 136.3,
129.4, 129.3, 129.1, 128.1, 128., 127.9, 79.8, 66.3, 62.1, 60.7,
59.2, 57.3, 55.8, 49.6, 45.4, 34.6, 34.0, 33.6, 30.1 (overlapping
with solvent signal), 29.8 (overlapping with solvent signal),
29.2, 28.6, 27.3, 22.2, 11.0; MS (ESI) m/z calcd for
[C52H70N5O11]+: 940.51 [M + H]+; found: 940.33.
2-((1S,5S,11S,15R)-18-(4-((4-((4-(((2-((3S,6S,7R,9aS)-3-(Benz-
hydrylcarbamoyl)-6-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-
butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepin-7-yl)ethyl)-
(tert-butoxycarbonyl)amino)methyl)benzyl)oxy)-4-oxobutanamido)-
methyl)benzyl)-11-(3-guanidinopropyl)-4,7,10,13,17,19-hexaoxo-
tR = 8.7 min; 1H NMR (400 MHz, CD3CN) δ 7.97 (d, J =
8.1 Hz, 1H), 7.86 (dd, J = 8.4, 3.6 Hz, 1H), 7.44–7.13 (m, 18H),
6.05 (d, J = 8.1 Hz, 1H), 5.08 (s, 2H), 4.99 (d, J = 15.2 Hz, 1H),
4.75 (t, J = 6.8 Hz, 1H), 4.54 (d, J = 9.4 Hz, 1H), 4.50 (dd, J = 7.9,
3,6,9,12,16,18-hexaazabicyclo[13.2.2]nonadecan-5-yl)acetic acid 4.2 Hz, 1H), 4.45 (dd, J = 9.0, 4.3 Hz, 1H), 4.31–4.23 (m, 3H),
21. Compound 19 (17 mg, 0.018 mmol, 1 eq.) was dissolved in
1 mL of DMF under nitrogen atmosphere in a flame dried
Schlenk tube. N-Hydroxysulfosuccinimide (5 mg, 0.023 mmol,
1.24 eq.) and N,N′-diisopropylcarbodiimide (4.18 μL,
0.027 mmol, 1.52 eq.) were then added at room temperature.
The solution was stirred overnight, then DMF was removed
under vacuum. The residue was then dissolved within the
same Schlenk tube in 1.2 mL of CH3CN, and a solution of
compound 2033 (12.4 mg, 0.0144 mmol, 0.8 eq.) in 1 mL of
phosphate buffer solution (the pH was adjusted to 7.5 with
0.2 M NaOH) was then added. The resulting mixture was
cooled to 0 °C, adjusting the pH to 7.5 with 0.2 M NaOH, and
stirring was continued overnight at room temperature. The
reaction mixture was then concentrated at reduced pressure
and purified by HPLC (gradient: from 90% H2O + 0.2%
HCOOH–10% acetonitrile + 0.2% HCOOH to 30% H2O + 0.2%
HCOOH–70% acetonitrile + 0.2% HCOOH in 15 minutes). The
purified product was then freeze-dried to give the desired pure
compound 21 as a white foam (9 mg, 0.072 mmol, 40% yield).
tR = 12.7 min; 1H NMR (400 MHz, CD3OD) δ 8.80 (d, J =
8.3 Hz, 1H), 8.40 (t, J = 5.5 Hz, 1H), 8.34–8.15 (br m, 1H),
7.39–7.12 (m, 18H), 6.12 (d, J = 8.3 Hz, 1H), 5.21–5.03 (m, 3H),
4.84–4.73 (m, 2H), 4.61 (t, J = 5.8 Hz, 1H), 4.58–4.27 (m, 7H),
4.24–4.15 (m, 1H), 4.07–3.91 (m, 3H), 3.88 (d, J = 6.3 Hz, 1H),
3.52 (d, J = 17.2 Hz, 1H), 3.45 (dd, J = 14.7, 6.8 Hz, 1H),
3.29–3.14 (m, 3H), 3.14–2.89 (m, 1H), 2.80 (s, 3H), 2.74–2.57
(m, 6H), 2.54 (t, J = 6.7 Hz, 2H), 2.30–2.16 (m, 1H), 2.15–1.33
(m, 33H), 0.92 (t, J = 7.2 Hz, 3H); 13C NMR (101 MHz, CD3OD)
δ 174.2, 174.0, 173.7, 173.6, 173.0, 172.9, 172.3, 172.2, 172.2,
171.3, 171.2, 171.1, 158.6, 143.1, 140.2, 136.7, 135.9, 135.9,
129.7, 129.5, 129.3, 128.7, 128.6, 128.5, 128.3, 81.4, 67.1, 62.8,
62.2, 60.6, 59.7, 58.3, 56.9, 54.3, 53.2, 51.3, 50.6, 48.1, 46.0,
4.10 (s, 2H), 4.04–3.89 (m, 3H), 3.89–3.78 (m, 3H), 3.46 (d, J =
17.1 Hz, 1H), 3.34 (dd, J = 15.0, 6.5 Hz, 1H), 3.12 (t, J = 6.5 Hz,
2H), 3.07–2.98 (m, 1H), 2.82–2.66 (m, 2H), 2.65–2.54 (m, 6H),
2.55–2.43 (m, 3H), 2.26–2.14 (m, 1H), 2.13–1.42 (m, 15H, over-
lapping with solvent signal), 0.92 (t, J = 7.5 Hz, 3H); 13C NMR
(101 MHz, CD3CN) δ 173.9, 173.2, 173.1, 173.0, 172.2, 171.2,
170.8, 170.6, 170.6, 170.0, 168.6, 168.5, 157.9, 143.1, 142.9,
140.1, 138.7, 135.4, 131.8, 131.2, 129.7, 129.3, 129.1, 128.8,
128.4, 128.3, 128.0, 66.4, 63.2, 62.4, 60.3, 59.2, 58.0, 56.5, 54.9,
52.8, 51.7, 50.0, 48.2, 46.4, 43.4, 43.2, 41.8, 39.8, 38.9, 37.3,
35.5, 33.6, 32.9, 32.5, 32.3, 31.1, 30.2, 29.0, 28.6, 26.9, 26.0,
23.9, 9.1; MS (ESI) m/z calcd for [C69H90N15O14]+: 1352.68 [M +
H]+; found: 1353.41; HRMS (ESI) m/z calcd for [C69H90N15O14]+:
1352.67862 [M + H]+; found: 1352.68112.
General procedure for the synthesis of compound 12. The
general procedure for the synthesis of compound 12 is
reported in the ESI.†
General procedure for the synthesis of compound 13
(3S,6S,7S,9aS)-Methyl 6-(tert-butoxycarbonylamino)-7-(hydroxy-
methyl)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate
32. Compound 3128 (1.06 g, 3.07 mmol, 1 eq.), ammonium
formate (2.23 g, 34.36 mmol, 11.2 eq.) and palladium hydrox-
ide on carbon (320 mg, 30%) were dissolved in a mixture of
THF–H2O 4 : 1 + 2% acetic acid (300 mL). The reaction mixture
was stirred at 70 °C for 6 h, then at rt overnight. The reaction
mixture was then filtered over celite, washing with THF and
CH2Cl2. The solvent was then removed at reduced pressure,
obtaining 1.12 g of crude aminoalcohol that was used without
any further purification.
A solution of Boc2O (1.73 g, 7.67 mmol, 2.5 eq.) in 10 mL of
dry CH2Cl2 was prepared and stirred at room temperature
under nitrogen atmosphere. The previously prepared crude
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aminoalcohol (theoretical 3.07 mmol, 1 eq.) and dry TEA under vigorous stirring. After 5 minutes a white solid started
(1.1 mL, 7.89 mmol, 2.6 eq.) in 20 mL of dry CH2Cl2 were then to precipitate. The solvent was then removed at reduced
added, and the reaction mixture was left under stirring at pressure, yielding 258 mg of desired pure compound 34 as a
room temperature for 20 h. The reaction mixture was then white solid (0.87 mmol, quantitative yield).
diluted with CH2Cl2 and washed with 2 × 30 mL of 5% citric
1H NMR (400 MHz, CDCl3) δ 4.58 (dd, 1H, J = 4.2 Hz, J =
acid solution, with 2 × 30 mL of saturated NaHCO3 and 2 × 8.3 Hz), 4.19 (d, 1H, J = 10.2 Hz), 4.01 (m, 1H), 3.72 (s, 3H),
30 mL of brine. The aqueous phase was washed with 2 × 2.80 (dd, 1H, J = 17.0 Hz, J = 3.8 Hz), 2.61 (dd, 1H, J = 17.0 Hz,
20 mL of CH2Cl2, the organic phases were dried with Na2SO4, J = 9.0 Hz), 2.32 (m, 1H), 2.26–2.14 (m, 2H), 2.11–2.02 (m, 1H),
filtered and the solvent was removed under reduced pressure. 2.01–1.92 (m, 2H), 1.92–1.74 (m, 3H). 13C NMR (100.6 MHz,
The crude product was purified by flash chromatography CDCl3) δ 170.1, 120.8, 70.4, 64.7, 64.2, 63.0, 62.0, 59.5, 55.5,
(n-hexane–AcOEt 6 : 4 as eluent), yielding 643 mg of pure com- 43.7, 40.7, 38.0, 37.4, 36.6, 36.1, 35.6, 35.3, 31.2, 31.1, 24.2. MS
pound 32 as a white solid (1.80 mmol, 59% yield over two (ESI): m/z calcd for C13H19N3O3: 265.14; found: 266.27
steps).
1H NMR (400 MHz, CDCl3) δ 6.14 (bd, 1H), 4.58 (dd, 1H, J =
[M + H+].
(3S,6S,7R,9aS)-Methyl 6-((R)-2-((tert-butoxycarbonyl)(methyl)-
amino)butanamido)-7-(cyanomethyl)-5-oxooctahydro-1H-pyrrolo-
[1,2-a]azepine-3-carboxylate 35. A solution of N-methyl-N-Boc-
aminobutyric acid (270 mg, 1.24 mmol, 1.4 eq.), EDC·HCl
(240.3 mg, 1.25 mmol, 1.4 eq.) and HOBt (181 mg, 1.34 mmol,
1.5 eq.) in 2.5 mL of dry CH2Cl2 was prepared under nitrogen
atmosphere, then 300 μL of dry DIPEA (1.68 mmol, 1.9 eq.)
were added. The solution was left under stirring for
10 minutes. A solution of compound 34 (258 mg, 0.87 mmol,
1 eq.) and 320 μL of dry DIPEA (1.79 mmol, 2.1 eq.) in 6.2 mL
4.6 Hz, J = 8.1), 4.25 (dd, 1H, J = 6.2 Hz, J = 10.5 Hz), 3.95–3.82
(m, 2H), 3.73 (s, 3H), 3.38 (dd, 1H, J = 12.1 Hz, J = 2.8 Hz), 2.25
(m, 1H), 2.18–1.96 (m, 4H), 1.92–1.72 (m, 3H), 1.55 (m, 1H),
1.42 (s, 9H). 13C NMR (100.6 MHz, CDCl3) δ 173.1, 171.4,
158.3, 81.3, 65.3, 61.3, 59.6, 55.9, 53.0, 42.5, 34.2, 33.5, 31.8,
28.9, 28.4. MS (ESI): m/z calcd for C17H28N2O6: 356.19; found:
257.0 [M − Boc + H]+.
(3S,6S,7R,9aS)-Methyl 6-((tert-butoxycarbonyl)amino)-7-(cyano-
methyl)-5-oxooctahydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate
33. A 0.1 M solution of compound 32 (210 mg, 0.59 mmol, of dry CH2Cl2 was then added dropwise, and the reaction
1 eq.) in dry CH2Cl2 (6 mL) was prepared and stirred under mixture was left under stirring at rt for 22 h. The reaction
nitrogen atmosphere; MsCl (101 mg, 0.88 mmol, 1.5 eq.) and mixture was diluted with CH2Cl2 and washed with 20 ml of a
dry TEA (246 μL, 1.76 mmol, 3 eq.) were added at 0 °C and the citric acid 5% solution, 20 mL of a NaHCO3 saturated solution
reaction mixture was then stirred for 2 h at room temperature. and 20 mL of brine. The organic phase was then dried with
After reaction completion the solution was diluted in 20 mL Na2SO4, filtered and the solvent was removed at reduced
of CH2Cl2, washed with a 5% citric acid aqueous solution pressure. The crude product was purified by flash chromato-
(2 × 50 mL), a saturated solution of NaHCO3 (2 × 50 mL) and graphy (n-hexane–EtOAc 4 : 6 as eluent), yielding 330 mg of
brine (2 × 50 mL). The organic phase was dried over Na2SO4 desired pure compound 35 as a white solid (0.71 mmol, 82%
and evaporated under reduced pressure. The crude mesylate yield).
(0.83 g, theoretical 1.91 mmol, quantitative yield) was used
without any purification.
1H NMR (400 MHz, CDCl3) δ 7.14 (bd, 1H), 4.59 (dd, 1H, J =
3.9 Hz, J = 7.3 Hz), 4.54 (m, 1H), 4.35 (m, 1H), 3.94 (m, 1H),
A 2 N solution of the crude mesylate in dry DMF (1 mL) was 3.76 (s, 3H), 2.85 (s, 3H), 2.64 (m, 1H), 2.38 (m, 1H), 2.34–2.26
+
prepared and nBu4 CN− (770 mg, 2.87 mmol, 1.5 eq.) was (m, 2H), 2.08 (m, 2H), 2.01 (m, 1H), 1.97–1.68 (m, 6H), 1.48
added. The solution was stirred at 80 °C in a microwave (s, 9H), 0.91 (t, 3H, J = 6.9 Hz). 13C NMR (100.6 MHz, CDCl3)
synthesizer for 2 h. The solvent was removed at reduced δ 173.1, 169.8, 79.8, 60.4, 58.7, 54.4, 52.5, 37.4, 33.8, 33.3, 32.6,
pressure and the crude product was purified by flash chrom- 28.2, 27.6, 21.4, 10.5. MS (ESI): m/z calcd for C23H36N4O6:
atography (n-hexane–EtOAc 4 : 6 as eluent). The desired pure 464.26; found: 465.54 [M + H+].
(3S,6S,7R,9aS)-Methyl 7-(2-aminoethyl)-6-((R)-2-((tert-butoxy-
carbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo-
[1,2-a]azepine-3-carboxylate 36. Hydrogenation of the nitrile 35
was performed using the H-Cube™ continuous-flow hydrogen-
ation reactor. Compound 35 (130 mg, 0.28 mmol, 1 eq.) was
dissolved in 50 mL of 90 : 10 EtOH–H2O and 1 M aqueous
citric acid (0.28 mmol) was added. The reaction mixture was
flowed through a Ni/Ra cartridge (hydrogen pressure: 60 bar;
T = 60 °C; flow 0.5 mL min−1). The reaction was completed
after 24 hours. The solvent was removed at reduced pressure.
The crude desired compound 36 (0.13 g, 0.277 mmol, quanti-
tative yield) was used for the next step without any
purification.
product 33 (0.59 g, 1.61 mmol) was obtained as a white
powder (yield 84%).
1H NMR (400 MHz, CDCl3) δ 5.83 (bd, 1H), 4.60 (dd, 1H, J =
7.9, J = 4.3 Hz), 4.26 (m, 1H, J = 10.0 Hz, J = 7.6 Hz), 3.93 (dd,
1H, J = 15.3 Hz, 7.6 Hz), 3.76 (s, 3H), 2.76 (dd, 1H, J = 4.5 Hz,
J = 17.0 Hz), 2.42–2.23 (m, 3H), 2.08 (m, 2H), 1.96 (m, 1H),
1.91–1.62 (m, 4H), 1.42 (s, 9H). 13C NMR (100.6 MHz, CDCl3)
δ 172.4, 169.6, 80.4, 60.6, 58.8, 56.3, 52.6, 37.8, 34.1, 33.5, 32.9,
28.4, 27.9, 21.0. MS (ESI): m/z calcd for C18H27N3O5: 365.20;
found: 366.2 [M + H]+.
(3S,6S,7R,9aS)-Methyl 6-amino-7-(cyanomethyl)-5-oxoocta-
hydro-1H-pyrrolo[1,2-a]azepine-3-carboxylate 34. Compound 33
(317 mg, 0.87 mmol, 1 eq.) was dissolved in 2.8 mL of dry
MeOH under nitrogen atmosphere at room temperature. Acetyl
chloride (930 μL, 13.03 mmol, 15 eq.) was added one pot and
MS (ESI): m/z calcd for C23H40N4O6: 468.29; found: 469.40
[M + H+].
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(3S,6S,7R,9aS)-Methyl 7-(2-benzamidoethyl)-6-((R)-2-((tert- filtered. The desired compound 40 (5.3 g, 24.6 mmol, quanti-
butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-
pyrrolo[1,2-a]azepine-3-carboxylate 37. Compound 36 (234 mg,
0.50 mmol, 1 eq.) was dissolved under inert atmosphere in
5.0 mL of dry CH2Cl2, then 350 μL of dry TEA were added
(2.50 mmol, 5 eq.). Benzoyl chloride (70 μL, 0.60 mmol,
1.2 eq.) was slowly added under vigorous stirring. The solution
tative yield) was obtained as a white powder and used for the
next step without any purification.
1H NMR (400 MHz, CDCl3) δ 8.25 (bs, 3H), 3.65 (s, 3H), 2.90
(m, 2H), 2.25 (t, 2H, J = 7.6 Hz), 1.70 (m, 2H), 1.60 (m, 2H),
1.40–1.15 (m, 12H).
(S)-Methyl 11-(2-((tert-butoxycarbonyl)amino)-2-phenylaceta-
turned from yellow to dark red. After 2 h additional TEA mido)undecanoate 41. A solution of (S)-N-Boc-phenylglycine
(50 μL, 0.37 mmol, 0.7 eq.) and benzoyl chloride (50 μL, (573 mg, 2.28 mmol, 1 eq.), HOBt (369 mg, 2.74 mmol,
0.43 mmol, 0.86 eq.) were added. After 3 h the reaction 1.2 eq.), EDC·HCl (524 mg, 2.74 mmol, 1.2 eq.) and dry DIPEA
mixture was diluted with CH2Cl2 and washed with 30 mL of a (1.39 mL, 7.98 mmol, 3.5 eq.) in dry CH2Cl2 (12 mL) was
5% citric acid solution, 30 mL of a saturated NaHCO3 solution stirred under nitrogen atmosphere for 15 minutes at 0 °C.
and 30 mL of brine. The organic phase was then dried with Then a solution of compound 40 (491 mg, 2.28 mmol, 1 eq.) in
Na2SO4, filtered and the solvent removed under reduced dry CH2Cl2 (12 mL) was slowly added, and the reaction
pressure. A brownish oil residue was purified by flash chrom- mixture was stirred at room temperature overnight. Then the
atography (n-hexane–EtOAc 1 : 9), yielding 264 mg of pure reaction mixture was diluted in 50 mL of CH2Cl2 and washed
desired compound 37 as a white solid (0.46 mmol, 92% yield).
with a 5% citric acid aqueous solution (2 × 50 mL), a saturated
1H NMR (400 MHz, [D6]acetone) δ 7.90 (m, 2H), 7.70 (bs, solution of NaHCO3 (2 × 50 mL) and brine (2 × 50 mL). The
1H), 7.54–7.42 (m, 3H), 7.16 (bs, 1H), 4.58–4.50 (m, 2H), 4.47 combined organic phases were dried over Na2SO4 and evapor-
(dd, 1H, J = 8.5 Hz, J = 3.9 Hz), 4.06 (m, 1H), 3.65 (s, 3H), 3.41 ated at reduced pressure. The residue was purified by flash
(m, 2H), 2.80 (m, 3H), 2.31 (m, 1H), 2.25–2.09 (m, 2H), chromatography (n-hexane–EtOAc 6 : 4 as eluent), and the
2.01–1.84 (m, 5H), 1.73 (m, 1H), 1.70–1.56 (m, 4H), 1.52 (m, desired pure product 41 (0.74 g, 1.65 mmol) was obtained as a
1H), 1.42 (s, 9H), 0.87 (t, 3H, J = 7.4 Hz). 13C NMR (100.6 MHz, white foam (yield 72%).
[D6]Acetone) δ 173.2, 171.4, 170.8, 167.0, 135.8, 131.3, 128.7,
1H NMR (400 MHz, CDCl3) δ 7.40–7.30 (m, 5H), 5.87 (bs,
127.6, 79.5, 60.2, 58.1, 55.0, 51.4, 37.4, 37.3, 33.0, 32.5, 31.7, 1H), 5.76 (bs, 1H), 5.13 (bs, 1H), 3.69 (s, 3H), 3.23 (dd, 2H, J =
27.5, 27.3, 21.3, 10.0. MS (ESI): m/z calcd for C30H44N4O7: 7.0 Hz, J = 13.1 Hz), 2.32 (t, 2H, J = 7.5 Hz), 1.63 (m, 2H), 1.43
572.32; found: 573.8 [M + H+].
(m, 9H), 1.38–1.10 (m, 14H). 13C NMR (100.6 MHz, CDCl3)
δ 174.3, 169.9, 155.2, 138.8, 129.0, 128.3, 127.2, 80.0, 58.7,
51.4, 39.8, 34.1, 29.3, 29.2, 29.1, 28.3, 26.6, 24.9. MS (ESI): m/z
calcd for C25H40N2O5: 448.29; found: 471.3 [M + Na+].
(S)-Methyl 11-(2-amino)-2-phenylacetamido)undecanoate 42.
Compound 41 (298 mg, 0.66 mmol, 1 eq.) was dissolved in a
3 N methanolic HCl solution (11 mL 33 mmol, 50 eq.) and
stirred under inert atmosphere at room temperature overnight.
The reaction was then quenched with 10 mL of saturated
NaHCO3 and extracted with EtOAc (3 × 10 mL). The combined
organic phases were dried over Na2SO4 and evaporated at
reduced pressure. The desired compound 42 was obtained
(0.22 g, 0.64 mmol, yield 97%) and used for the next step
without any purification.
(3S,6S,7R,9aS)-7-(2-Benzamidoethyl)-6-((R)-2-((tert-butoxy-
carbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-pyrrolo-
[1,2-a]azepine-3-carboxylic acid 30. A 0.1 M solution of com-
pound 37 (35 mg, 0.061 mmol, 1 eq.) in 1 mL of THF–H2O 3/1
was prepared at 0 °C, and 2 N aqueous LiOH (92 μL,
0.183 mmol, 3 eq.) was slowly added. The reaction mixture was
stirred at 0 °C for 30 minutes, then at room temperature for
5 h. The reaction was then quenched with 1 mL of 1 M
aqueous KHSO4 and the resulting mixture was extracted with
CH2Cl2 (3 × 5 mL). The combined organic phases were dried
over Na2SO4 and evaporated at reduced pressure. The crude
desired product 30 (0.03 g, 0.054 mmol, yield 88%) was used
for the next step without any purification.
1H NMR (400 MHz, CDCl3) δ 7.83 (m, 2H), 7.68 (bd, 1H),
7.48–7.32 (m, 4H), 4.76 (m, 1H), 4.57 (m, 1H), 4.51 (m, 1H),
3.99 (m, 1H), 3.41 (m, 2H), 2.94 (bs, 3H), 2.26 (m, 1H),
2.14–1.99 (m, 4H), 1.91–1.71 (m, 5H), 1.65–1.51 (m, 3H), 1.37
(s, 9H), 0.94 (t, 3H, J = 6.0 Hz). 13C NMR (100.6 MHz, CDCl3)
δ 174.2, 173.0, 127.6, 135.3, 131.5, 128.8, 128.7, 128.1, 127.7,
81.1, 60.7, 60.1, 58.6, 54.7, 37.2, 36.7, 32.8, 32.7, 32.5, 31.4,
30.7, 29.5, 28.1, 27.4, 22.7, 10.8. MS (ESI): m/z calcd for
C29H42N4O7: 558.31; found: 559.7 [M + H+].
1H NMR (400 MHz, CDCl3) δ 7.42–7.26 (m, 5H), 7.03 (bs,
1H), 4.53 (s, 1H), 3.68 (s, 3H), 3.26 (ddd, 2H, J = 13.1 Hz, J =
7.1 Hz, J = 2.2 Hz), 2.31 (t, 2H, J = 7.5 Hz), 1.35–1.23 (m, 12H).
13C NMR (100.6 MHz, CDCl3) δ 175.0, 173.5, 141.7, 129.3,
128.4, 127.3, 60.0, 51.4, 39.2, 34.0, 29.4, 29.2, 29.1, 29.0, 26.7,
24.8. MS (ESI): m/z calcd for C20H32N2O3: 348.24; found: 349.6
[M + H+].
Methyl 11-((S)-2-((3S,6S,7R,9aS)-7-(2-benzamidoethyl)-6-((R)-2-
((tert-butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-
11-Aminoundecanoic acid methyl ester 40. A 0.5 M solution of 1H-pyrrolo[1,2-a]azepine-3-carboxamido)-2-phenylacetamido)-
undecanoate 43. Compound 30 (45.0 mg, 0.081 mmol, 1 eq.),
HATU (47.0 mg, 0.124 mmol, 1.4 eq.), HOAt (24.3 mg,
0.178 mmol, 2 eq.) and dry DIPEA (50 μL, 0.28 mmol, 3 eq.)
were dissolved in dry DMF (0.6 mL) under nitrogen atmos-
phere at room temperature. The solution was left under
stirring for 10 minutes. Then a solution of compound 42
11-aminoundecanoic acid 39 (5.09 g, 25.3 mmol, 1 eq.) in
MeOH (50 mL) was prepared and SOCl2 (2.93 mL, 40.4 mmol,
1.6 eq.) was slowly added. The reaction mixture was stirred at
room temperature for 2 h, then refluxed at 80 °C for additional
2 h. The solvent was then evaporated at reduced pressure
and the solid residue was washed with 10 mL of dry Et2O, then
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(46.8 mg, 0.122 mmol, 1.4 eq.) and dry DIPEA (100 μL, 7.67 (m, 0.5H), 7.50–7.18 (m, 8H), 5.53 (d, 0.5H, J = 7.7 Hz),
0.56 mmol, 6 eq.) in dry DMF (0.6 mL) was prepared under 5.50 (d, 0.5H, J = 7.8 Hz), 4.76 (m, 1H), 4.69 (m, 1H), 4.20 (m,
nitrogen atmosphere, and was added dropwise. The resulting 1H), 4.10 (m, 0.5H), 4.03 (m, 0.5H), 3.61 (s, 3H), 3.44 (m, 2H),
reaction mixture was left under stirring at rt for 43 hours. 3.15 (m, 2H), 2.78 (s, 3H), 2.28 (td, 2H, J = 7.4 Hz, J = 1.8 Hz),
Then, the solvent was removed under reduced pressure, the 2.22 (m, 1H), 2.19–2.09 (m, 2.5H), 2.05 (m, 0.5H), 2.02–1.94
crude was dissolved in EtOAc (90 mL) and washed with a 5% (m, 2.5H), 1.91 (m, 0.5H), 1.90–1.83 (m, 2.5H), 1.76–1.62 (m,
citric acid aqueous solution (25 mL) and a saturated NaHCO3 2.5H), 1.57 (m, 2H), 1.43 (m, 2H), 1.33 (m, 1H), 1.31–1.19 (m,
solution (25 mL). The organic phase was then dried with 12H), 1.03 (m, 3H). 13C NMR (100.6 MHz, [D6]acetone,
Na2SO4, filtered and the solvent was removed at reduced ≈1 : 1 mixture of two conformers) δ 130.4, 128.0, 127.6, 127.3,
pressure. The crude product (100 mg) was purified by flash 127.2, 126.8, 126.7, 61.0, 60.4, 57.3, 56.1, 54.6, 49.6, 38.1, 36.4,
chromatography (EtOAc 100% as eluant), and the desired pure 36.1, 35.7, 35.6, 32.7, 32.6, 32.5, 32.4, 32.1, 31.9, 31.8, 31.7,
product 43 (44.1 mg, 0.050 mmol, yield 61%) was obtained as 31.3, 29.3, 28.3, 28.2, 26.2, 26.1, 25.5, 25.4, 23.7, 21.1, 21.0, 7.1.
Compound 46. Compound 38 (31.5 mg, 0.036 mmol, 1 eq.),
HATU (20.8 mg, 0.053 mmol, 1.5 eq.), HOAt (10.0 mg,
0.073 mmol, 2 eq.) and dry DIPEA (50 μL, 0.28 mmol, 7.8 eq.)
were dissolved in dry DMF (200 μL) and stirred for 10 minutes
at room temperature under nitrogen atmosphere. Then a solu-
tion of compound 4516 (40.8 mg, 0.051 mmol, 1.4 eq.) in dry
DMF (300 μL) was added dropwise. The reaction mixture was
left stirring at room temperature for 72 h. The solvent was
thoroughly removed under reduced pressure, then the crude
was re-dissolved in EtOAc (50 mL) and washed with a 5% citric
acid solution (30 mL) and with a saturated NaHCO3 solution
(30 mL). The organic phase was then dried over Na2SO4, fil-
tered and the solvent was removed under reduced pressure.
The crude residue was purified by flash chromatography
(Biotage™, reverse phase, from 5% CH3CN–95% H2O to 100%
CH3CN as eluent), yielding pure 46 as a white solid (18.1 mg,
0.011 mmol, 30% yield).
a white solid.
1H NMR (400 MHz, [D6]DMSO, ≈1 : 1 mixture of two confor-
mers) δ 8.61 (bd, 0.5H), 8.48 (d, 0.5H, J = 8 Hz), 8.33 (m, 1H),
8.21 (m, 0.5H), 7.88–7.72 (m, 3H), 7.68 (m, 0.5H), 7.52–7.21
(m, 8H), 5.36 (d, 0.5H, J = 6 Hz), 5.34 (d, 0.5H, J = 5.9 Hz), 4.59
(dd, 1H, J = 7.4 Hz, 3.7 Hz), 4.52 (m, 1H), 4.40 (m, 1H),
4.00–3.90 (m, 1H), 3.58 (s, 3H), 3.23 (m, 2H), 3.04 (m, 2H), 2.70
(s, 1.5H), 2.69 (s, 1.5H), 2.28 (td, 2H, J = 7.4 Hz, 2.8 Hz), 2.14
(m, 1H), 2.05 (m, 1H), 1.94 (m, 1H), 1.86 (m, 1H), 1.80 (m, 1H),
1.79–1.70 (m, 2H), 1.68–1.57 (m, 2H), 1.57–1.38 (m, 6H), 1.35
(m, 2H), 1.34 (s, 9H), 1.26–1.14 (m, 12H), 0.81 (bt, 3H). 13C
NMR (100.6 MHz, [D6]Acetone, ≈1 : 1 mixture of two confor-
mers) δ 172.2, 170.2, 170.5, 170.1, 169.9, 167.0, 140.1, 139.5,
135.8, 131.3, 129.0, 128.8, 128.7, 128.6, 128.2, 128.1, 127.8,
127.7, 127.6, 61.2, 61.1, 58.3, 57.2, 57.0, 54.9, 39.1, 37.3, 34.1,
33.4, 33.1, 32.9, 32.7, 31.9, 27.6, 26.4, 26.3, 24.7, 10.0. MS
(ESI): m/z calcd for C49H72N6O9: 889.13; found: 890.0 [M + H+].
1H NMR (400 MHz, [D6]acetone, mixture of two conformers)
δ 8.51–8.30 (m, 1H), 8.16–8.06 (m, 1H), 7.92–7.44 (m, 4H), 7.59
(bs, 1H), 7.51–7.17 (m, 9H), 6.96 (m, 1H), 6.68 (s, 1H), 6.51 (bs,
11-((S)-2-((3S,6S,7R,9aS)-7-(2-Benzamidoethyl)-6-((R)-2-((tert-
butoxycarbonyl)(methyl)amino)butanamido)-5-oxooctahydro-1H-
pyrrolo[1,2-a]azepine-3-carboxamido)-2-phenylacetamido)undecanoic
acid 38. Compound 43 (34.1 mg, 0.038 mmol, 1 eq.) was dis- 1H), 5.44 (m, 1H), 4.68 (m, 1H), 4.59 (m, 1H), 4.57 (m, 1H),
solved in THF–H2O 3 : 1 (400 μL), and 2 N aqueous LiOH 4.53 (m, 1H), 4.30 (m, 1H), 4.26 (m, 1H), 4.20 (m, 1H), 4.14 (m,
(60 μL, 0.122 mmol, 3 eq.) was slowly added. The reaction 1H), 4.09 (m, 0.5H), 4.00 (m, 0.5H), 3.84 (s, 3H), 3.47–3.25 (m,
mixture was stirred at room temperature for 5 h. Then the reac- 3H), 3.23–3.06 (m, 8H), 2.99 (m, 1H), 2.80 (s, 3H), 2.72 (m,
tion mixture was quenched with 1 M aqueous KHSO4 (1 mL), 1H), 2.67 (s, 3H), 2.61 (s, 3H), 2.55 (m, 1H), 2.44–2.31 (m, 3H),
diluted with distilled water (10 mL) and extracted sequentially 2.26 (m, 1H), 2.14 (m, 1H), 2.08 (m, 2H), 2.05 (m, 2H),
with CH2Cl2 (3 × 15 mL) and EtOAc (3 × 15 mL). The combined 2.02–1.85 (m, 4H), 1.75–1.18 (m, 52H), 0.88 (m, 3H). 13C NMR
organic phases were dried over Na2SO4 and evaporated at (75.4 MHz, [D6]acetone, mixture of two conformers) δ 174.7,
reduced pressure. The desired compound 38 (0.0315 g, 172.2, 171.0, 170.8, 158.8, 157.3, 137.0, 135.9, 131.7, 129.3,
0.036 mmol, yield 95%) was obtained as a white solid and 129.0, 128.5, 128.0, 124.7, 112.4, 80.9, 63.3, 62.0, 59.1, 57.8,
used for the next step without any purification. MS (ESI): m/z 56.6, 55.8, 54.3, 52.0, 43.6, 41.1, 40.0, 38.1, 37.4, 36.8, 35.8,
calcd for C48H70N6O9: 874.52; found: 875.8 [M + H+].
34.2, 33.6, 32.8, 30.5, 30.3, 29.8, 29.3, 29.0, 28.5, 28.2, 27.3,
26.8, 26.3, 24.2, 22.4, 18.6, 14.3, 12.1, 11.1, 8.0. MS (ESI): m/z
calcd for C84H123N15O18S: 1662.89; found: 1685.9 [M + Na+].
Cyclo-RGD/SMAC mimetic conjugate 13. Compound 46
(16.1 mg, 0.0097 mmol, 1 eq.) was dissolved in TFA–thioani-
sole–1,2-ethanedithiol–anisole 90 : 5 : 3 : 2 (2 mL) and stirred at
room temperature for 2 h. After reaction completion, the
solvent was evaporated under reduced pressure, then the
crude was dissolved in H2O (10 mL) and washed with iPr2O
(10 mL). The aqueous phase was evaporated under reduced
pressure and the crude purified by HPLC (Waters Atlantis Prep
T3 OBD 5 µm, 19 × 100 mm, column; solvents: (A) H2O + 0.1%
TFA, (B) CH3CN + 0.1% TFA gradient from 90%A–10%B to
Methyl 11-((S)-2-((3S,6S,7R,9aS)-7-(2-benzamidoethyl)-6-((S)-2-
(methylamino)butanamido)-5-oxooctahydro-1H-pyrrolo[1,2-a]-
azepine-3-carboxamido)-2-phenylacetamido)undecanoate
44.
Compound 43 (5.3 mg, 0.0060 mmol, 1 eq.) was dissolved
under inert atmosphere in 3 N methanolic HCl (200 μL). The
reaction mixture was left under vigorous stirring at room temp-
erature for 4 hours, then the solvent was slowly removed under
reduced pressure. The pure desired acid 44 (5.0 mg,
0.0060 mmol, quantitative yield) was obtained as a white solid.
1H NMR (400 MHz, [D6]acetone, ≈1 : 1 mixture of two con-
formers) δ 8.64 (m, 0.5H), 8.57 (m, 0.5H), 8.53 8 m, 0.5H), 8.45
(m, 0.5H), 8.21 (m, 0.5H), 8.16–8.03 (m, 2.5H), 7.89 (m, 0.5H),
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30%A–70% B over 15 min.; flow rate 15 mL min−1, λ = 210 nm, with 100 μL per well Substrate Reagent Solution (R&D Systems,
Rt = 8.8 min). The purified product was then freeze-dried to Minneapolis, MN, USA) before stopping the reaction with the
give the trifluoroacetate salt of the desired pure compound 13 addition of 50 μL per well 2 N H2SO4. Absorbance at 415 nm
as a white foam (8.4 mg, 0.0059 mmol, 57% yield).
was read in a Synergy™ HT Multi-Detection Microplate Reader
1H NMR (400 MHz, D2O) δ: 7.66 (d, 2H), 7.52 (m, 1H), 7.42 (BioTek Instruments, Inc.). Each data point represents the
(q, 2H, J = 7.8 Hz) 7.38–7.28 (m, 5H), 5.23 (m, 1H), 4.62 (dd, average of triplicate wells; data analysis was carried out by non-
1H, J = 9.3 Hz, J = 7.3 Hz), 4.55–4.40 (m, 3H), 4.33 (d, 1H, J = linear regression analysis with GraphPad Prism software. Each
8.4 Hz), 4.25–4.15 (m, 2H), 4.06 (m, 1H), 3.98–3.83 (m, 2H), experiment was repeated in triplicate.
3.48–3.29 (m, 3H), 3.24–2.95 (m, 7H), 2.76–2.63 (m, 2H), 2.62
General procedure for the measurement of cytotoxic activity.
(s, 3H), 2.60 (s, 3H), 2.42–2.30 (m, 3H), 2.21 (m, 1H), 2.15–1.97 Human breast MDA-MB231 and ovarian IGROV-1 carcinoma
(m, 5H), 1.96–1.74 (m, 7H), 1.72–1.50 (m, 6H), 1.49–1.39 (m, cell lines were cultured in vitro with RPMI plus 10% fetal calf
4H), 1.34 (m, 2H), 1.22–1.11 (m, 5H), 1.10–0.99 (m, 8H), 0.95 serum. For cytotoxic experiments, 104 cells were plated in 96
(t, 3H, J = 7.5 Hz), 0.90 (t, 3H, J = 7.5 Hz). 13C NMR (75.5 MHz, well plates and grown overnight before being treated with
D2O) δ: 178.3, 175.2, 174.6, 173.8, 172.3, 171.5, 171.0, 136.9, serial dilutions of the compounds (concentrations ranging
132.9, 129.9, 129.6, 127.9, 127.8, 63.6, 62.9, 62.5, 59.0, 57.3, from 25 µM to 10 nM). Cell viability was determined after
57.1, 56.7, 53.7, 52.3, 43.2, 41.2, 40.9, 40.1, 38.3, 35.8, 33.6, 72 hours using the CellTiter-Glo (Promega) assay for ATP.
33.0, 32.3, 31.7, 30.9, 29.2, 29.1, 28.3, 27.7, 26.5, 26.1, 25.2, Three independent experiments were performed in triplicates.
24.3, 9.0. HRMS (ESI): m/z calcd for C65H95N15O13: 1294.7301;
found: 1294.73243 [M + H+].
Acknowledgements
Biology
General procedure for the fluorescence polarization-based We thank Milan University for PhD Fellowships (to M.M. and
BIR/IAP binding assay. The affinity of each tested compound A.D.C.). We also gratefully acknowledge “Ministero dell’Univer-
for the BIR domains of XIAP and cIAP2 was evaluated as pre- sità e della Ricerca” for financial support (PRIN project
viously described.36–38 Recombinant XIAP (60 nM final con- 2010NRREPL: Synthesis and biomedical applications of
centration) and cIAP2 (25 nM) BIR3, or XIAP BIR23 (3 nM) tumour-targeting peptidomimetics).
domains were diluted in the assay buffer containing 100 mM
potassium phosphate, pH 7.5, 100 µg mL−1 bovine γ-globulin
and 0.02% sodium azide. Serial dilutions of each tested com-
pound (concentrations ranging from 2 µM to 0.2 nM) were
Notes and references
added to displace a fluorescent probe (FITC-SMAC for BIR3
domains; SMAC-1F for BIR23) and fluorescence polarization
was measured with the Ultra plate reader (Tecan), at excitation
and emission wavelengths of 485 nm and 530 nm, respectively.
All experiments were performed in black, flat-bottom 96 well
plates (Greiner Bio-One). Three independent experiments were
performed in duplicates. The analyses were performed using
Graphpad Prism v.5.02.
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