Homo- and heterodimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part I: Synthesis

Article abstract of DOI:10.1016/j.bmc.2012.09.020

Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures connected through a head-head (8), tail-tail (9) or head-tail (10) linker. The selection of appropriate decorating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The synthesis, purification and analytical characterization of each prepared dimer 8-10 is thoroughly described.

Full text of DOI:10.1016/j.bmc.2012.09.020

Bioorganic & Medicinal Chemistry 20 (2012) 6687–6708
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Homo- and heterodimeric Smac mimetics/IAP inhibitors as in vivo-active
pro-apoptotic agents. Part I: Synthesis
Leonardo Manzoni a, Laura Belvisi b, Aldo Bianchi c, Annalisa Conti d, Carmelo Drago c,
Marilenia de Matteo c, Luca Ferrante c, Eloise Mastrangelo e, Paola Perego d, Donatella Potenza b,
Carlo Scolastico c, Federica Servida f, Gabriele Timpano c, Francesca Vasile b, Vincenzo Rizzo c,
Pierfausto Seneci b,c,
⇑
a Istituto di Scienze e Tecnologie Molecolari (ISTM), Consiglio Nazionale delle Ricerche (CNR), Via Golgi 19, Milan I-20133, Italy
b Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, Milan I-20133, Italy
c CISI scrl, Via Fantoli 16/15, Milan I-20138, Italy
d Dipartimento di Oncologia Sperimentale e Medicina Molecolare, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, Milan I-20133, Italy
e Dipartimento di Bioscienze e CNR-IBF, Università degli Studi di Milano, Via Celoria 26, Milan I-20133, Italy
f Fondazione Matarelli, Dipartimento di Farmacologia, Chemioterapia e Tossicologia Medica, Università degli Studi di Milano, Via Vanvitelli 32, Milan I-20129, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 21 September 2012
Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like
4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures con-
nected through a head–head (8), tail–tail (9) or head–tail (10) linker. The selection of appropriate deco-
rating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The
synthesis, purification and analytical characterization of each prepared dimer 8–10 is thoroughly
described.
Keywords:
IAP inhibitors
Smac mimetics
Apoptosis
Ó 2012 Elsevier Ltd. All rights reserved.
Oncology
Peptidomimetics
1. Introduction
for pro-apoptotic drugs in oncology.18 The initial assumption of a
common anti-apoptotic mechanism for the whole IAP target family
The apoptotic process of programmed cell death is dysfunc-
tional in its regulation in a variety of human pathologies, among
which cancer,1–3 inflammation4,5 and neurodegeneration,6,7 where
it has become the focus of extensive pharmaceutical research.
Pathways leading to apoptosis include the extrinsic or death recep-
tor-dependent path8,9 and the intrinsic or mitochondrial path,8,10
both caspase-dependent; and some caspase-independent mecha-
nisms, involving other proteases as apoptosis executioners.11
In a complex scenario, hitting more than one putative signifi-
cant target with a lead should maximize the chances of therapeutic
success. Thus, we focused on targeting molecular entities involved
in several apoptotic pathways, rather than extremely targeted
agents. Such polypharmacology12 approach is precedented in
target classes such as kinases13 and GPCRs.14
through interference with caspase-dependent pathways19,20 has
been proven wrong, or at least only partially correct for other IAP
proteins,21,22 but their interest as oncology targets related to apop-
tosis has not been seriously challenged.23
The most caspase-connected IAP is the X-Inhibitor of Apoptosis
Protein24,25 (XIAP). XIAP is capable of binding caspase 9 (the initia-
tor caspase) and both caspases 3 and 7 (the executioner caspases).26
XIAP binding prevents activation of caspases and, consequently,
prevents cells from entering apoptosis.
A protein released from mitochondria, the Second Mitochon-
dria-derived Activator of Caspases (Smac)27/Direct IAP Binding
protein with LOw pI (DIABLO)28 binds XIAP as a dimer29 on the
same binding sites of caspase 9 (BIR3 domain).30–32 Smac interferes
also with the XIAP binding site of caspases 3 and 7 (linker-BIR2 do-
main),33 promoting both the extrinsic and intrinsic apoptosis
paths. The delicate balance of this binding equilibrium is altered
in various diseases; for example, several tumor cell lines show
overexpression of XIAP and, consequently, a caspase-dependent
resistance to enter apoptosis.34,35 Thus, XIAP inhibition via Smac
The family of Inhibitor of Apoptosis Proteins (IAPs)15,16 is char-
acterized by the presence of one or more Baculovirus IAP Repeat
(BIR) domains. Since the discovery of the first viral iap gene and
its linkage with apoptosis,17 IAPs were considered putative targets
mimetics’ binding is considered
a validated mechanism for
⇑
Corresponding author. Tel.: +39 0250314060; fax: +39 0250314075.
E-mail address: pierfausto.seneci@unimi.it (P. Seneci).
intervention in cancer therapy.36–38
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.020

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L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
Smac binding to other human IAP family members such as
cIAP139 and cIAP240 through their BIR domains is also proven,
homodimers (9), together with unprecedented heterodimeric
head–tail compounds (10, Fig. 3). We aimed to introduce symmetri-
cal and unsymmetrical substitution patterns on the two linker-con-
nected monomer units, and to assess a few synthetic strategies fully
compatible with a wide variety of functional groups. We reasoned
that, if fully achieved, such goals should have allowed to understand
the influence of structural modifications on in vitro and in vivo effi-
cacy of dimeric Smac mimetics/IAP inhibitors 8–10, and to select at
least one suitable candidate for preclinical evaluation.
The rational design and the chemical synthesis of dimeric Smac
mimetics/IAP inhibitors 8–10 is presented and discussed in this pa-
per. Their structural and biological characterization, including
NMR and X-ray studies, cell-free, cellular and in vivo efficacy test-
ing on selected compounds, are reported in the following paper.58
although not related to their NF-jB activation-related anti-apopto-
tic properties.41–43 Thus, Smac mimetics may modulate the
pathological action of any IAP oncology target,44–46 although the
complex network of IAP-dependent interactions still needs to be
fully elucidated to understand any drawback for the use of Smac
mimetics in therapy.47
Several authors have shown how monomeric Smac mimetics/
IAP inhibitors such as 1a (Fig. 1, top left), inspired by the Smac
N-terminal AVPI sequence and built on aza-containing bicycloal-
kane carboxylate scaffolds, bind XIAP, cIAP1 and cIAP2 on their
BIR domains with sub-micromolar potency, induce rapid cIAP1
degradation, and affect apoptosis in tumor cells when given in
combination with cytotoxic agents.48 The same authors reported
diazabicyclic scaffolds as potent, orally active pan-IAP antago-
nists.49 Among them, 2a (AT406, Fig. 1, bottom left) is in clinical
development for the treatment of solid and hematological tu-
mors.50 Recently, another orally active clinical candidate 3a
(GDC-0152, Fig. 1, top right) was fully described.51
We have reported a structure-driven approach to potent, mono-
meric Smac mimetics/IAP inhibitors of general formula 4 (Fig. 1,
middle), where introduction of a 4-substitution on the 1-aza-2-
oxobicyclo[5.3.0]decane scaffold established novel molecular
interactions with the binding linker-BIR2 and/or BIR3 sites.52,53
Our early lead 4a52 (Fig. 1, bottom right) showed good in vitro—
cell-free and cellular—potency. The optimized lead 4b54 (Fig. 1,
bottom right) showed a significant improvement in solubility and
penetration through biological membranes.
2. Synthesis
2.1. Rationale
Some of us recently reported52,59 an innovative synthesis strat-
egy to access monomeric compounds of general formula 4, which
differ from known structures 1 for the additional 4-substitution
on the 1-aza-2-oxobicyclo[5.3.0]decane scaffold (Fig. 1). Having se-
cured synthetic access to such structures, and having established a
preliminary SAR on the 4-position, we decided to study how two
such structures could be connected, through a suitable linker at-
tached onto a chemically modifiable functional group, to take
advantage of a simultaneous interaction with two BIR domains,
as previously reported by others.55,57
Dimeric Smac mimetics/IAP inhibitors such as 5–7 (Fig. 2) were
also presented as bifunctional ligands able to simultaneously bind
toBIR2andBIR3domainsofXIAP, andtoinducerapidcIAP1andcIAP2
degradation.55–57 Their structure includes two AVPI-related pepti-
domimetics connected through one (5, 7) or two (6) linkers of varying
length, rigidity and lipophilicity. The observed cytotoxic effects on se-
lected cell lines55 and significant activity in animal models57 sug-
gested a downstream developability for such ‘non drug-like’ dimers.
Here we report novel, dimeric Smac mimetics/IAP inhibitors
8–10 (Fig. 3) which take advantage of their characteristic 4-substitu-
tion to exploit three connection strategies. Labeling as ‘head’ the
4-substituent and ‘tail’ the C-terminus amide of the 1-aza-2-oxobi-
cyclo[5.3.0]decane scaffold (Fig. 3), we have designed, synthesized
and biologically characterized several head–head (8) and tail–tail
As to the connecting chemical function between the monomer
unit and the linker, it has to be both chemically replaceable-modi-
fiable, and compatible with preserving BIR-binding properties even
after substantial structural modifications. Two such groups were
known at the time when we designed our dimer assembly strategy:
the C-terminus amide portion, used in several papers as an anchor
point for dimer synthesis,55,57 and the 4-substitution, which was
proven by us to tolerate the introduction of various functional
groups, including bulky substituents.52 We named the latter com-
pounds as head–head dimers 8, while the former were labelled as
tail–tail compounds 9.
We reasoned that, in order to reach both binding sites in two
BIR domains of a given IAP protein, the two monomeric AVPI
mimetics would need to adopt specific—and possibly rather
H
N
N
O
N
H
N
O
O
S
N
N
( )n
4
3
X
N
H
5
6
NH
8
10
7
O
O
1
N
H
9
2
O
N
HN
AA
N
H
1a
R
O
3a
Y
N
H
O
X heteroatom,
4
=
AA aminoacid,
=
H
N
R alkyl subst.
=
O
N
H
N
N
N
H
N
O
N
H
O
O
O
2a
NH
O
NH
O
Y H
4a
4b
=
N
Y CF
=
3
H
Figure 1. Monomeric Smac mimetics/IAP inhibitors.

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6689
HN
NH
O
O
N
O
N
O
N
H
N
H
H
N
O
N
H
O
O
O
N
O
N
H
N
N
N
N
N
O
S
S
N
N
N
NH
6
5
HN
O
O
H
N
N
H
N
N
H
O
N
O
N
HN
N
O
O
(CH2)4
N
(H2C)4
N
H
N
N
N
NH
H
N
HN
O
O
H
N
7
SM-164
O
O
N
Figure 2. Previously reported dimeric Smac mimetics/IAP inhibitors.
8
H
( )n
4
X
HEAD-HEAD CONNECTION
N
Ph2HC
N
H
HEAD
T
10
N
O
N
O
CHPh2
TAIL
R
HN
AA
O
N
T
O
( )n
O
X
HN
N
H
O
AA
LINK
( )n
X
NH
AA
O
H
N
O
CHPh2
T
LINK
N
O
N
N
H
H
O
N
N
O
O
O
N
LINK
H
N
( )n
X
NH
O
HN
AA
NH
AA
AA
H
H
( )n
( )n
NH
AA
10
HEAD-TAIL CONNECTION
X
X
H
( )n
9
X
TAIL-TAIL CONNECTION
Figure 3. Head–head (8), tail–tail (9) and head–tail (10) dimeric Smac mimetics/IAP inhibitors based on the 1-aza-2-oxobicyclo[5.3.0]decane scaffold.
constrained—conformations. A graphically effective pictorial anal-
ogy of head–head, head–tail and tail–tail dimers is reported in
Figure 4. It is likely that, to assume a relative orientation of both
monomers in a given dimer 8 or 9 which would allow to bind
simultaneously to two BIR targets, either one or both of the linked
monomers should move away from their minimum energy state.
It’s also reasonable that their capability to reach such dimer bind-
ing conformation would be different, due to the constraints in-
duced in dimer regions by a tail-tail or a head-head connection.
We then reasoned that, if synthetically feasible, head-tail heterodi-
mers 10 would also provide useful information in terms of binding
modes, and obviously of dimers’ structural requirements to
maximize their pro-apoptotic effects (Fig. 4).
mimetics. We selected several linking backbones, taking also
advantage of reported efforts by other groups,60 with the aim to ex-
plore how, inter alia, their length, flexibility and lipophilicity would
influence the dimer activity. Some preferred linker structures are
depicted in Figure 5.
2.2. Key monomers
Six monomeric intermediates were selected as synthetic gate-
ways to our dimers. Their structure is shown in Figure 6, while
their synthesis was previously reported.52
Namely, compound 4c corresponds to the product of step ii,
Scheme 1, Ref. 52; compound 4d corresponds to the product of
step iv, Scheme 1, Ref. 52; compounds 4e,f and 4g,h correspond,
respectively, to compounds 8a,b and 9a,b, Scheme 1 in Ref. 52.
The linker unit, connecting two AVPI mimetics, is also going to
influence the bi-functional binding potential of dimeric Smac

6690
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
8
H
N
HEAD-HEAD CONNECTION
Ph HC
2
N
H
T
N
O
CHPh
2
O
N
T
O
( )n
X
HN
O
LINK
AA
( )n
X
NH
AA
O
O
LINK
N
H
N
H
N
N
O
O
H
N
HN
AA
NH
H
CHPh2
H
( )n
( )n
AA
O
N
T
X
X
O
9
O
N
H
LINK
TAIL-TAIL CONNECTION
N
( )n
X
NH
O
AA
NH
10
HEAD-TAIL CONNECTION
H
( )n
AA
X
Figure 4. Heads, tails and swallows.
HO
HO
O
N
( )n
H
N
H
N
N
N
BOC
N
N
( )n
N
N
N
O
( )n
N
H2N
N
H
O
O
O
O
OMe
O
OH
4d
4c
N
N
H
N
H
N
N
H
N
H
N
ht
H2N
O
N
ht
n
3
O
( )n
O
( )n
BOC
n
O
BOC
N
O
O
O
O
O
e,g
f,h
R
R
=
=
H
Me
N
O
R
N
R
N
N
H
N
H
O
O
O
O
NH
NH
4e,f
( )n
4g,h
( )n
( )n
O
O
O
O
( )n
Figure 6. Key monomeric synthons for the synthesis of dimers 8–10.
Figure 5. Preferred linker structures.
N-Methylamine 4h was reacted with three biscarboxylates
(step a, Scheme 3) to provide good yields of N-Boc-protected di-
mers 12d–f. Acidic deprotection with TFA (step b) provided good
to excellent yields of head–head dimers 8d–f as bistrifluoroace-
tates (Scheme 3). The unoptimized overall yields for 8d, 8e and
8f were, respectively, 44%, 53% and 39%.
2.3. Head–head homodimers 8
Nine head–head homodimers 8 were synthesized. A diphe-
nylmethylamide was the tail for all compounds 8, while three of
them contained (S)-ethylglycine and six contained N-methyl(S)-
ethylglycine (AA, Fig. 3).
N-Methylamine 4h was also reacted with an alkynyl heptanoate
(step a, Scheme 4) to provide N-methyl, 4-alkynyl amide 11c in
excellent yield. This was oxidatively dimerized55 in excellent yields
(step b) using copper (II) acetate, and the resulting symmetrical N-
Boc-protected dimer 12g was deprotected (step c) to provide mod-
erate yields of head–head dimer 8g as a dihydrochloride (Scheme
4). The unoptimized overall yield for 8g was 30%.
Amine 4g was reacted with two alkynyl carboxylic acids (step a,
Scheme 1) to provide high yields of 4-alkynyl amides 11a,b. The
amides were reacted with azide 4e in a Cu-catalyzed click chemis-
try reaction (step b, Scheme 1) to provide good yields of N-Boc-pro-
tected dimers 12a,b. Acidic deprotection with methanolic HCl (step
c) provided high yields of head–head dimers 8a,b as bishydrochlo-
rides (Scheme 1). The unoptimized overall yields for 8a and 8b
were, respectively, 38% and 42%.
The amide 11c was also reacted with a known bis-azide61 in a
Cu-catalyzed click chemistry reaction (step a, Scheme 5) to provide
moderate yields of the N-Boc-protected dimer 12h. Acidic depro-
tection with TFA (step b) provided quantitative yields of the
head–head dimer 8h as a bistrifluoroacetate (Scheme 5). The unop-
timized overall yield for 8h was 29%.
Alkynylamide 11b was oxidatively dimerized55 in moderate
yields (step a, Scheme 2) using copper (II) acetate, and the resulting
symmetrical N-Boc-protected dimer 12c was deprotected (step b)
to provide quantitative yields of head–head dimer 8c as a dihydro-
chloride (Scheme 2). The unoptimized overall yield for 8c was 36%.

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6691
H
O
( )n
4g
N
H
+
O
BOC
HN
a
b
N
O
N
H
H
O
COOH
( )n
NH
11a - 87%
11b - 85%
a
b
n = 0
n = 2
H
N
BOC
H
O
N
O
N
N
O
N
N
O
N
c
b
H
N
O
H
N
N
H
( )n
BOC
O
12a - 47%
12b - 54%
O
N
H
N
H
.HCl
NH2
H
O
N
O
c
N
O
N
O
N
H
N
N
N
H
( )n
N
O
N
H
O
O
N
H
8a - 92%
8b - 90%
H2N
.HCl
a, DCC, DMAP, CH2Cl2, 0°C to rt, 1 hr; b, 2d, Cu(OAc)2, Na ascorbate, t-BuOH:water 1:1, rt, 18 hrs;
c, 3N HCl in MeOH, rt, 18 hrs.
Scheme 1. Synthesis of head–head dimers 8a,b.
O
a
11b
N
O
H
N
N
H
O
H
N
BOC
O
N
H
BOC
H
N
N
H
O
O
N
b
O
H
N
N
H
12c - 36%
O
O
b
N
O
H
N
N
H
O
.HCl
NH2
O
N
H
H
N
H2N
.HCl
O
O
N
O
H
N
H
N
8c - quant.
O
a, Cu(OAc)2, CH3CN, reflux, 0.5 hrs; b, 3N HCl in MeOH, rt, 18 hrs.
Scheme 2. Synthesis of head–head dimer 8c.
Finally, N-methylamine 4h was reacted with N-Cbz beta alanine
(step a, Scheme 6) to provide N-methyl amide 11d in quantitative
yield. The amide was hydrogenolytically deprotected (step b) and
reacted with diethyl squarate (step c) to give the symmetrical,
N-Boc-protected dimer 12i in excellent yield. Acidic deprotection
with TFA (step d) provided excellent yields of the head–head dimer
8i as a bistrifluoroacetate (Scheme 6). The unoptimized overall
yield for 8i was 75%.

6692
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
HOOC
LINK COOH
4h +
d
e
f
link = (CH2)6
link = (OCH2CH2)2O
a
link = (OCH2CH2)3O
N
H
N
BOC
O
O
N
O
N
O
b
N
H
O
H
N
LINK
N
O
H
N
H
O
12d - 68%
12e - 56%
12f - 52%
O
N
H
BOC
N
H
.TFA
N
H
O
N
O
O
O
N
O
H
N
LINK
H
N
H
N
N
H
b
N
O
8d - 64%
8e - 95%
8f - 75%
O
O
N
H
N
.TFA
H
a, EDC, HOBt, DIPEA, CH2Cl2, rt, 2 hrs; b, TFA, CH2Cl2, rt, 4 hrs.
Scheme 3. Synthesis of head–head dimers 8d–f.
N
BOC
H
a
N
O
4h
O
N
O
H
N
N
H
( )3
O
b
O
11c - 92%
N
H
( )3
BOC
N
H
N
O
O
O
N
H
N
H
N
BOC
N
O
O
N
O
H
N
H
( )3
N
12g - 84%
O
O
c
N
O
H
N
( )3
N
H
O
H
O
N
H
.TFA
N
H
N
N
H
.TFA
O
O
N
O
H
N
N
H
( )3
8g - 41%
O
a, 6-heptynoic acid, EDC, HOBt, DIPEA, CH2Cl2, rt, 4 hrs; b, Cu(OAc)2, CH3CN, reflux, 0.5 hrs;
c, TFA, CH2Cl2, rt, 4 hrs.
Scheme 4. Synthesis of head–head dimer 8g.
2.4. Tail–tail homodimers 9
in good yield. The amide was reacted with a known bis-azide58 in a
Cu-catalyzed click chemistry reaction (step b) to provide the
N-Boc-protected dimer 14a in good yield. Acidic deprotection with
TFA (step c) provided tail–tail dimer 9a as bistrifluoroacetate in
good yield (Scheme 7). The unoptimized overall yield for 9a was
29%.
A set of four tail–tail dimers 9b–e was prepared by reaction of
the same carboxylic acid 4d with four bis-amine linkers 17a–d.
Such bis-amines were prepared from bis-amines 15a–d62 by
Seven tail–tail homodimers 9 were synthesized. N-methyl(S)-
ethylglycine was present in all compounds 9 (AA, Fig. 3), while
three different 4-substitutions (–CH2OH in five compounds, -
CH2NHCH2Ph and elongated –CH2CH2NH2 in one compound) were
introduced.
Carboxylic acid 4d was reacted with an optically active alkynyl
amine (step a, Scheme 7) to provide C-terminus-alkynyl amide 13a

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6693
O
( )4
a
N3
N
O
H
N
( )3
N
H
+
11c
N3
O
( )4
N
O
BOC
N
H
N
N
N
b
( )4
12h - 32%
( )4
N
N
N
N
BOC
O
H
N
O
O
N
O
H
N
( )3
.TFA
N
H
O
N
H
N
H
O
( )3
N
N
O
N
H
H
N
O
N
N
( )4
b
( )4
8h - 92%
N
N
N
H
H
N
.TFA
N
O
O
N
O
a, Cu(OAc)2, Na ascorbate, t-BuOH:water 1:1, rt, 18 hrs;
b, TFA, CH2Cl2, rt, 4 hrs.
H
N
N
H
( )3
O
Scheme 5. Synthesis of head–head dimer 8h.
O
N
CBzHN
a
H
N
O
4h
BOC
H
N
N
H
O
N
O
11d - quant.
b,c
N
BOC
N
H
N
H
N
O
O
O
BOC
O
N
O
O
N
N
O
N
O
H
H
N
N
H
H
( )2
( )2
N
N
12i - 92%
H
H
O
N
O
d
N
H
.TFA
( )2
.TFA
( )2
H
N
H
H
N
O
O
O
O
N
O
N
O
N
O
O
H
H
N
N
H
H
N
N
H
N
8i - 83%
H
O
O
a, N-CBz beta-alanine, EDC, HOBt, DIPEA, CH2Cl2, rt, 2 hrs; b, Pd/C, H2, continuous flow, 10 bar, 40°C; c, diethyl
squarate, TEA, EtOH, DMAP, rt, 18 hrs; d,TFA, CH2Cl2, rt, 4 hrs.
Scheme 6. Synthesis of head–head dimer 8i.
condensation with (S)-phenylglycine (step a, Scheme 8) to give
N-Boc protected compounds 16a–d in excellent yields. Desired
bis-amine linkers 17a–d were obtained by TFA deprotection (step
b) in quantitative yields (Scheme 8).
Carboxylic acid 4d was reacted with four bis-amine linkers
17a–d (step a, Scheme 9) to give, with good to excellent yields,
N-Boc protected dimers 14b–e. Acidic deprotection with TFA (step
b) provided tail–tail dimers 9b–e as bistrifluoroacetates in good to
excellent yields (Scheme 9). The unoptimized overall yields for
9b–e, starting from 4d and 17a–d, were, respectively, 53%, 73%,
31% and 56%.
N-Boc protected dimer 14a was decorated on its 4-substitution
to provide tail–tail dimer 9f (Scheme 10). Namely, 14a was mesy-
lated and reacted with sodium azide (steps a, b) to give azide 14f in
good yield. The azide was reduced in a Staudinger reaction (step c)
to give the amine 14g in quantitative yield, and the amine was
N-alkylated in a reductive amination protocol (step d) to provide
the N-protected, benzylated dimer 14h in moderate yields. Acidic

6694
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
HO
4d
( )4
O
BOC
N
3
a
+
b
N
O
+
N
N
N
H
3
( )4
O
N
H
13a - 67%
NH2
H
HO
O
N
BOC
O
( )4
N
N
O
N
N
N
H
N
H
N
N
N
N
O
14a - 74%
N
( )4
b
N
O
O
OH
BOC
N
H
c
H
N
.TFA
HO
O
H
N
O
( )4
N
O
N
N
N
H
N
H
N
N
N
N
.TFA
O
N
( )4
9a - 59%
N
O
O
OH
H
N
H
a, EDC, HOBt, DIPEA, dry CH2Cl2, rt, 2 hrs; b, Cu(OAc)2, Na ascorbate, tBuOH:water 1:1, rt, 18 hrs;
c,TFA, CH2Cl2, rt, 4 hrs.
Scheme 7. Synthesis of tail–tail dimer 9a.
b
H
N
H
N
H
N
H
N
LINK
LINK
a
H2N
NH2
.TFA
HN
BOC
NH
15a-d
.TFA
BOC
O
O
O
O
16a - 94%
16b - 94%
16c - 88%
16d - 83%
17a-d - quant.
a, N-Boc-(S)-PheGly, HOBt, HBTU, collidine,
dry DMF, rt, 2 hrs; b, TFA, CH2Cl2, rt, 4 hrs.
H2N
O
O
H2N
NH2
NH2
O
15b
15a
15c
O
O
O
O
H2N
NH2
H2N
N
H
N
H
N
N
15d
H
H
NH2
Scheme 8. Synthesis of bis-amine linkers 17a–d.
deprotection with TFA (step e) provided tail–tail dimer 9f as a
tetratrifluoroacetate in good yield (Scheme 10). The unoptimized
overall yield for 9f from 14a was 24%.
tive basic hydrolysis of the methyl ester (step g), the acid 13e was
reacted with previously described bis-amine 17b (step h) to give
tetra N-Boc protected dimer 14i in good yields. Acidic deprotection
with TFA (step i) provided elongated tail–tail dimer 9g as a tetratri-
fluoroacetate in good yield (Scheme 11). The unoptimized overall
yield for 9g was 12%.
Finally, 4-hydroxymethyl methyl ester 4c was N-Boc protected,
mesylated and reacted with a tetraalkyl ammonium cyanide (steps
a–c, Scheme 11) to provide the cyano compound 13b in good yield.
After N-deprotection and condensation with N-protected and
methylated (S)-ethylglycine (steps d,e) to yield 13c, the cyano
group was reduced, and the resulting amine was protected with
Boc in a one-pot reaction protocol (steps f,a) to give the N-Boc
protected, elongated amine 13d in moderate yield. After quantita-
2.5. Head–tail heterodimers 10
Three head–tail heterodimers 10 were synthesized together
with a hybrid heterodimeric structure. N-Methyl(S)-ethylglycine

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6695
H
O
N
H
N
O
H
N
LINK
H
N
O
O
N
H
N
O
N
BOC
N
a
N
H
4d
BOC
O
HO
O
O
OH
N
b
14b - 97%, 14c - 80%,
14d - 51%, 14e - 74%
H
N
O
N
H
O
H
N
LINK
H
N
b
O
O
N
H
.TFA
H
N
O
N
N
N
H
H
O
O
.TFA
O
N
HO
9b - 58%, 9c - 97%,
9d - 69%, 9e - 91%
OH
a, 17a-d, HOBt, HBTU, collidine, dry DMF, 0° to rt, 2 hrs; b, TFA, CH2Cl2, rt, 4 hrs.
Scheme 9. Synthesis of tail–tail dimers 9b–e.
14a
H
N
a,b
N3
O
BOC
O
( )4
N
N
O
N
N
N
H
N
H
N
N
N
N
14f - 58%
O
N
( )4
N
O
O
N3
BOC
N
H
H
c
H2N
N
BOC
O
O
( )4
N
N
O
N
N
N
N
H
N
H
N
N
N
O
14g - quant.
N
( )4
N
O
O
NH2
BOC
N
H
H
d
N
HN
BOC
O
O
( )4
N
N
O
N
N
N
N
H
N
H
N
N
N
O
14h - 45%
N
( )4
N
O
O
NH
BOC
N
H
e
H
HN
O
N
H
N
O
( )4
N
O
N
.TFA
N
N
H
N
H
N
N
N
N
.TFA
O
N
9f - 92%
( )4
N
H
O
O
NH
N
H
a, MsCl, TEA, dry CH2Cl2, 0° to rt, 2 hrs; b, NaN3, dry DMF, 100° C, 0.5hrs; c, Me3P, dry CH2Cl2, rt,
2 hrs; d, PhCHO, NaBH(OAc)3, dry CH2Cl2, rt, 18 hrs; e, TFA, CH2Cl2, rt, 4 hrs.
Scheme 10. Synthesis of tail–tail dimer 9f.

OR
13d - 44
, R
=Me
%
13e - quant., R
=H
6696
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
H
4c
BOC
N
NC
a-c
O
O
BOC
BOC
NC
N
O
N
O
N
N
f,a
OMe
N
H
N
H
d,e
O
O
N
O
BOC
N
H
13c - 67%
O
OMe
g
13b - 63%
h
H
O
N
H
N
O
H
N
LINK
H
N
O
O
N
H
N
N
O
N
N
H
BOC
O
14i - 77%
BOC
O
O
N
BOC
N
BOC
N
i
H
H
H
N
O
N
O
H
H
LINK
H
N
N
O
O
N
H
.TFA
H
N
N
H
O
N
N
O
O
9g - 80%
H
.TFA
O
N
.TFA
H2N
.TFA
NH2
a, Boc2O, TEA, CH2Cl2, rt, 2 hrs; b, MsCl, TEA, CH2Cl2, rt, 2 hrs; c, nBu4N+CN-, DMF, microwave,
80°C, 1.5 hrs; d, 3N HCl in MeOH, rt, 18 hrs; e, N-Boc,N-Me-(S)-2aminobutanoic acid, EDC,
HOBt, DIPEA, dry CH2Cl2, rt, 2 hrs; f, Ni Raney, EtOH, continuous flow, 60 bar, 60°C, 6 hrs;
g, 2N LiOH, 1,4-dioxane:water, 0°C to rt, 1 hr; h, 17b, HOBt, HBTU, collidine, dry DMF, rt,
18 hrs; i, TFA, CH2Cl2, rt, 4 hrs.
Scheme 11. Synthesis of tail–tail dimer 9g.
(AA, Fig. 3), 4-CH2OH substitution, and a diphenylmethyl amide
tail were conserved in the three heterodimers, while the hybrid
structure connected the head of a 1-aza-2-oxobicyclo[5.3.0]decane
scaffold-based AVPI mimetic with a structurally different, known55
monomeric unit.
Four key tail-linker monomeric intermediates 18a–d were pre-
pared as shown in Scheme 12. Commercially available azidoamine
15e was first reacted with (S)-phenylglycine(step a) to give interme-
diate 16e in good yield, then N-deprotected (step b). Coupling with
4d (step c) provided the linker-bearing monomeric azide 18a in
excellent yield. After catalytic hydrogenation (step d) of the same
azide to provide quantitatively the linker-bearing monomeric amine
18b, the same amine was either coupled with 4-pentynyl-butanoic
acid (step e) to give the linker-bearing monomeric alkyne 18c with
excellent yield, or with diglycolic anhydride (step f) to give the
linker-bearing monomeric carboxylate 18d (Scheme 12).
Linker-bearing carboxylate 18d was coupled with amine 4h
(step a, Scheme 15) to provide in very good yield the N-protected
head–tail heterodimer 19c. Acidic deprotection with TFA (step b)
provided head–tail heterodimer 10c as a bistrifluoroacetate in
excellent yield (Scheme 15). The unoptimized overall yield for
10c was 59% from 18d.
Finally, a heterodimer containing two linker-connected AVPI
mimetics based on different scaffolds was prepared as in Scheme
16. Namely, azide 4e was coupled in a click chemistry reaction
with N-protected alkyne-containing Smac mimetic 20a55 (step a)
to give N-Boc, N-Fmoc protected heterodimer 19d in good yield.
Sequential deprotection in basic (step b) and acidic (step c) condi-
tions produced the hybrid heterodimer 10d in excellent yield. The
unoptimized overall yield for 10d was 51%.
3. Conclusions
Linker-bearing monomeric azide 18a was coupled in a click
chemistry reaction with 4-alkynyl amide 11e (step b, Scheme
13), this last prepared from 4h (step a), to provide in good yield
the N-protected head–tail heterodimer 19a. Acidic deprotection
with TFA (step c) provided head–tail heterodimer 10a as a bistri-
fluoroacetate in excellent yield (Scheme 13). The unoptimized
overall yield for 10a was 55% from 18a.
The chemical feasibility of head head-8, tail tail-9 and head tail-
10 dimers based on 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane
scaffolds was successfully proven through the synthesis of 20 com-
pounds, bearing various 4-substitutions. The monomer units in
each dimer were connected through linkers with varying length,
lipophilicity and flexibility.
Linker-bearing alkyne 18c was coupled in a click chemistry
reaction with azide 4f (step a, Scheme 14) to provide in very good
yield the N-protected head–tail heterodimer 19b. Acidic deprotec-
tion with TFA (step b) provided head–tail heterodimer 10b as a bis-
trifluoroacetate in excellent yield (Scheme 14). The unoptimized
overall yield for 10b was 69% from 18c.
Extensive details regarding the biological activity of each pre-
pared dimer, and the structural characterization of selected homo-
dimers using NMR, X-ray crystallography and gel filtration are
provided in the following paper.58 Their activity profiling will drive
our future synthetic efforts towards the more prospective homo-
and heterodimers. Such efforts will be reported in due time.

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6697
BOC
NH
O
O
O
H2N
N
O
N
H
3
O
O
15e
a
b,c
16e - 74%
O
N
3
HO
O
BOC
b,c
N
O
N
N
H
H
N
N
O
3
O
O
O
N
H
O
18a - 82%
d
HO
O
BOC
N
O
N
N
H
H
N
O
NH2
O
O
O
N
H
O
18b - 98%
O
O
HO
O
BOC
e
NH
N
O
18b
N
N
H
H
N
O
O
O
O
O
O
N
H
O
18c - 74%
HO
HOOC
O
O
BOC
f
18b
NH
N
N
N
H
N
H
O
O
N
H
O
O
18d - 58%
a, N-Boc-(S)-phenylglycine, HOBt, HBTU, collidine, dry DMF, 0° to rt, 2 hrs; b, TFA, CH2Cl2, rt,
4 hrs; c, 4d, HOBt, HBTU, collidine, dry DMF, 0° to rt, 2 hrs; d, H2, Pd/C, continuous flow,
10 bar, 35°C; e, 4-pentynoic acid, DCC, DMAP, dry CH2Cl2, 0° to rt, 4 hrs; f, diglycolic
anhydride, pyridine, dry DMF, rt, 4 hrs.
Scheme 12. Synthesis of tail-linker monomeric intermediates 18a–d.
Solvents were distilled and dried according to standard proce-
dures, and reactions requiring anhydrous conditions were per-
formed under nitrogen or argon. Solvents for the reactions were
used directly from the bottle if not differently specified.
Standard dimers 355 and 557 were prepared according to the
published procedure.
4. Experimental part
4.1. General methods
1H NMR spectra were recorded on a Bruker Avance instrument
in CDCl3, CD3OD or D2O as solvent at 400 or 600 MHz. 13C NMR
spectra were recorded in CDCl3, CD3OD or D2O as solvent at 100
or 125 MHz. Coupling constants are given in Hertz, Hz, and are
rounded to the nearest 0.1 Hz.
4.2. Head–head dimer 8a—Scheme 1
4.2.1. Compound 11a
Purifications were carried out either by flash chromatography on
A solution of DCC (20.6 mg, 0.10 mmol) and DMAP (2.4 mg,
0.02 mmol) in dry CH2Cl2 (10 mL) was added dropwise to a stirred
solution of compound 4g (59.2 mg, 0.10 mmol) and propiolic acid
silica gel (particle size 60 lm, 230–400 mesh), Kieselgel, or by
Biotage™ flash chromatography [Biotage columns Si-12-M (150 ꢀ
12 mm; silica gel (40–63 lm), flow rate 12 mL/min); Si-25-M
(6.2 lL, 0.10 mmol) in dry CH2Cl2 (10 mL) at 0 °C. After the addition,
(150 ꢀ 25 mm; silica gel (40–63
Biotage™ C18 reverse phase chromatography [Biotage column
m), flow rate 25 mL/
min)]. Final products were purified by C18 reverse phase semi-pre-
parative HPLC using either a Waters X-Terra RP18 OBD column
lm), flow rate 25 mL/min)], or by
the reaction mixture was warmed to room temperature over a per-
iod of 1 h. After reaction completion, the reaction mixture was fil-
tered, and the filtrate was washed with diethyl ether (2 ꢀ 10 mL).
The combined organic phase was concentrated under reduced pres-
sure, and the crude product was purified by Biotage™ flash chroma-
tography, eluant conditions: 1% of MeOH and 99% of CH2Cl2 to 10% of
MeOH and 90% of CH2Cl2. Yield 85% (55 mg, MW 644.32,
C
18HS (150 ꢀ 25 mm; KP-C18-HS (35–70
l
(19 mm ꢀ 10.0 cm,
5
l
l
m) or
m).
a Supelco Ascentis C18 column
(21.2 mm ꢀ 15.0 cm, 5
LC–MS data were collected with an Agilent 1100 HPLC con-
nected to a Bruker Esquire 3000+ ion trap mass spectrometer
through an ES interface.
0.085 mmol) of pure 11a. Analytical characterization: ½a 2D0
ꢂ81.4
ꢁ
(c 0.75, MeOH); 1 H NMR (400 MHz, CDCl3 ): d: 7.91 (d, J = 8.8 Hz,
1H), 7.50–7.10 (m, 11H), 6.24 (d, J = 8.4 Hz, 1H), 5.00 (d, J = 6.4 Hz,
1H), 4.75 (d, J = 7.2 Hz, 1H), 4.64 (t, J = 9.2 Hz, 1H), 4.20 (dd,
Optical rotations ½a 2D0
ꢁ
were measured in cells of 1 dm path-
length and 1 mL capacity with a Perkin Elmer 241 polarimeter.

6698
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
4h
a
N
BOC
H
N
O
O
N
O
N
O
N
HN
H
N
H
O
H
N
b
N
O
BOC
11e - 93%
O
H
N
HO
O
O
BOC
19a - 61%
N
O
N
N
H
H
N
N
O
N
O
N
O
c
O
N
H
O
O
N
HN
O
H
N
a, 4.pentynoic acid, EDC, HOBt, DIPEA, CH2Cl2, rt,
4 hrs; b, 18a, Cu(OAc)2, Na ascorbate,
tBuOH:water 1:1, rt, 18 hrs; c, TFA, CH2Cl2, rt, 4 hrs.
N
H
O
H
N
.TFA
HO
O
O
10a - 90%
H
N
O
N
N
H
H
N
N
.TFA
O
N
O
N
O
O
N
H
O
Scheme 13. Synthesis of head–tail dimer 10a.
171.3, 169.5, 155.5, 152.2, 142.1, 141.1, 128.7, 127.3, 80.2, 74.6, 68.0,
61.0, 58.7, 56.8, 53.0, 39.1, 34.2, 33.3, 31.3, 28.3, 25.6, 10.4.
N
H
N
O
O
BOC
N
O
N
H
4.2.2. Compound 12a
a
N
4f
N
A 0.9 M water solution of sodium ascorbate (45
and a 0.3 M water solution of Cu(OAc)2 (65 L, 0.02 mmol) were
sequentially added to stirred solution of compounds 4e
(61.8 mg, 0.10 mmol) and 11a (64.4 mg, 0.10 mmol) in a 1:1 mix-
ture of H2O/tBuOH (300
L). The reaction mixture was stirred over-
lL, 0.4 mmol)
N
l
O
a
HO
BOC
19b - 80%
O
NH
N
O
N
l
N
H
H
N
O
night at room temperature, and then the solvent was removed
under reduced pressure. Finally, the residue was purified by Bio-
tage™ flash chromatography, eluant conditions: 1% of MeOH and
99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 47%
(59 mg, MW 1261.54, 0.047 mmol) of pure 11a. Analytical charac-
O
O
b
O
N
H
O
N
H
terization: ½a D20
ꢁ
ꢂ64.3 (c 0.51, CHCl3); 1H NMR (400 MHz, CDCl3): d:
H
O
O
.TFA
N
N
7.82 (d, J = 14.0, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.32–7.00 (m, 24H),
6.12 (t, J = 8 Hz, 2H), 5.07 (d, J = 5.6 Hz, 1H), 4.91 (d, J = 11.2 Hz,
1H), 4.64 (t, J = 6.4 Hz, 2H), 4.55 (m, 3H), 4.14 (t, J = 11.6 Hz, 1H),
3.98 (m, 2H), 3.73 (m, 3H), 3.14 (d, J = 12.0 Hz, 1H), 2.34 (m, 2H),
2.16 (m, 2H), 2.0–1.40 (m, 16H), 1.31 (s, 18H), 1.19 (s, 2H), 0.90
(m, 6H); 13C NMR (100 MHz, CDCl3): d: 173.1, 171.7, 170.6,
169.4, 169.2, 160.4, 142.1, 141.8, 141.0, 128.8, 128.7, 127.7,
127.5, 127.4, 127.2, 127.1, 61.3, 61.1, 58.8, 56.8, 56.7, 54.0, 53.7,
40.7, 40.6, 40.2, 34.4, 33.8, 33.3, 33.2, 31.4, 25.7, 25.5, 25.4, 10.3.
N
O
N
H
N
N
O
HO
O
10b - 86%
H
N
.TFA
NH
N
N
H
N
H
O
O
O
O
N
H
O
O
a,18c, Cu(OAc)2, Na ascorbate, tBuOH:water 1:1, rt, 18 hrs; b, TFA, CH2Cl2, rt, 4 hrs.
4.2.3. Compound 8a
Scheme 14. Synthesis of head–tail dimer 10b.
A 3 N solution of HCl in MeOH (0.5 mL) was added to a stirred
solution of compound 12a (7.6 mg, 0.006 mmol) in MeOH (2 mL).
The reaction mixture was left stirring at room temperature
overnight and then concentrated under reduced pressure. The
J = 10.8, 7.2 Hz, 1H), 4.06 (m, 2H), 3.85 (q, J = 8.4 Hz, 1H), 2.95 (s, 1H),
2.45 (m, 1H), 2.28 (m, 1H), 2.0–1.55 (m, 7H), 1.47 (s, 9H), 1.35–1.10
(m, 2H), 0.97 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 172.4,

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6699
(dd, J = 13.6, 8.4 Hz, 1H), 3.28 (dd, J = 12.8, 8.4 Hz,1H), 2.25–1.35
(m, 22H), 0.95 (m, 6H); 13C NMR (100 MHz, D2O): d: 172.7, 170.5,
169.8, 169.7, 161.9, 142.2, 141.0, 140.9, 129.0, 128.9, 127.8,
127.4, 127.1, 61.9, 58.6, 58.4, 57.6, 54.5, 54.4, 54.3, 41.0, 38.6,
38.1, 32.4, 32.3, 27.8, 24.4, 8.5, 8.4. ESI-MS: m/z 1061.5
N
BOC
H
N
O
O
N
H
a
4h
O
H
N
N
O
[M+H]+1083.6 [M+Na]+, 531.3 [M+2H]2+
.
O
4.3. Head–head dimer 8b—Scheme 1
O
HO
19c - 67%
O
BOC
NH
The head–head dimer 8b was prepared similarly to 8a. The de-
tailed synthetic protocol leading to its synthesis, and its full syn-
thetic characterization are reported in the Supplementary data.
N
O
N
N
H
H
N
O
O
O
O
N
H
O
b
4.4. Head–head dimer 8c—Scheme 2
N
H
.TFA
4.4.1. Compound 12c
O
O
H
N
N
H
A stirred suspension of compound 11b (67.2 mg, 0.10 mmol)
and Cu(OAc)2 (127.1 mg, 0.70 mmol) in acetonitrile (20 mL) was
refluxed for 30 min After reaction completion, the solvent was re-
moved under reduced pressure, the residue was dissolved in
CH2Cl2 and Cu(II) salts were removed by filtering over a short
pad of silica gel eluting with 9/1 CH2Cl2/MeOH. Finally, the crude
products was purified by Biotage™ flash chromatography, eluant
conditions: 1% of MeOH and 99% of CH2Cl2 to 10% of MeOH and
90% of CH2Cl2. Yield 36% (48 mg, MW 1341.67, 0.036 mmol) of
O
H
N
N
O
O
O
HO
O
10c - 88%
H
N
NH
N
O
N
H
H
.TFA
N
O
O
O
O
N
H
pure 12c. Analytical characterization: ½a 2D0
ꢁ
ꢂ69.3 (c 0.96, CHCl3);
O
1H NMR (400 MHz, CDCl3): d: 7.90 (d, J = 6.8 Hz, 2H), 7.36–7.16
(m, 25H), 6.22 (d, J = 8.0 Hz, 1H), 5.05 (bd, J = 6.4 Hz, 2H), 4.72 (d,
J = 8.0 Hz, 2H), 4.49 (t, J = 9.6 Hz, 2H), 3.98 (dd, J = 13.6, 7.6 Hz,
2H), 3.81 (dd, J = 17.6, 8.8, 2H), 3.58 (m, 2H), 3.01 (m, 1H), 2.58
(m, 4H), 2.40 (m, 6H), 2.25 (m, 2H), 1.95–1.55 (m, 16H), 1.45 (s,
18H), 1.30–1.10 (m, 2H), 1.00 (t, J = 7.6 Hz, 6H); 13C NMR13C
NMR (100 MHz, CDCl3): d: 173.6, 171.7, 170.8, 169.4, 141.2,
140.8, 128.6, 127.5, 127.4, 127.2, 80.5, 66.0, 61.2, 58.9, 57.0, 56.8,
a,18d, HOBt, HBTU, collidine, dry DMF, 0° to rt, 18 hrs; b, TFA, CH2Cl2, rt, 4 hrs.
Scheme 15. Synthesis of head–tail dimer 10c.
residue did not require any further purification, and was lyophi-
lized. Yield 92% (6 mg, MW 1134.22, 0.0055 mmol) of pure 8a.
Analytical characterization: ½a 2D0
ꢁ
ꢂ39.1 ( c 0.23, H2O); 1H NMR
(400 MHz, D2O): d: 8.35 (s, 1H), 7.32–7.18 (m, 20H), 5.97 (m,
2H), 4.61 (m, 3H), 4.48 (m, 2H), 4.35 (m, 1H), 3.9 (m, 4H), 3.50
O
N
N
N
a
N
N
O
4e
+
O
N
H
S
20a
N
Fmoc
H
N
BOC
N
N
N
HN
N
N
O
N
O
O
O
a
N
N
b,c
S
N
N
H
O
O
NH
19d - 57%
N
Fmoc
NH2
N
N
N
N
HN
N
N
N
O
O
b,c
O
O
N
S
N
N
H
O
O
NH
10d - 90%
N
H
a, Cu(OAc)2, Na ascorbate, t-BuOH:water 1:1, rt, 18 hrs; b, piperidine, DMF, rt, 0.5 hrs;
c, 3N HCl in MeOH, rt, 18 hrs.
Scheme 16. Synthesis of head–tail dimer 10d.

6700
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
54.0, 41.3, 40.0, 35.3, 34.5, 33.2, 28.3, 25.7, 25.2, 15.9, 10.3. ESI-MS:
m/z 1342.9 [M+H]+, 1363.9 [M+Na]+.
31.7, 31.0, 30.0, 28.9, 27.5, 25.6, 23.3, 8.0. ESI-MS: m/z 1177.6
[M+H]+, 589.2 [M+2H]2+
.
4.6. Head–head dimers 8e,f—Scheme 3
4.4.2. Compound 8c
A 3 N solution of HCl in MeOH (0.5 mL) was added to a stirred
solution of compound 12c (18.5 mg, 0.014 mmol) in MeOH
(2 mL). The reaction mixture was left stirring at room temperature
overnight and then concentrated under reduced pressure. The res-
idue did not require any further purification, and was lyophilized.
Quantitative yield (17 mg, MW 1214.35, 0.014 mmol) of pure 8c.
The head–head dimers 8e and 8f were prepared similarly to 8d.
The detailed synthetic protocols leading to their synthesis, and
their full synthetic characterization are reported in the Supplemen-
tary data.
4.7. Head–head dimer 8g—Scheme 4
Analytical characterization: ½a 2D0
ꢁ
ꢂ43.1 ( c 0.90, H2O); 1H NMR
(400 MHz, D2O): d: 7.35–7.20 (m, 20H), 5.95 (s, 2H), 4.56 (d,
J = 10.4 Hz, 2H), 4.50 (dd, J = 7.6, 4.8 Hz, 2H), 3.98 (m, 4H), 3.23
(dd, J = 13.6, 3.2 Hz, 2H), 3.06 (m, 2H), 2.45–2.30 (m, 4H), 2.25–
1.40 (m, 26H), 0.97 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, D2O):
d: 174.3, 172.4, 170.4, 169.6, 141.1, 140.9, 129.0, 128.9, 127.9,
127.8, 127.4, 127.0, 65.8, 61.7, 58.4, 57.7, 54.5, 54.4, 40.7, 37.6,
34.3, 32.6, 30.7, 27.8, 24.4, 15.5, 8.5. ESI-MS: m/z 1163.3
4.7.1. Compound 11c
EDCꢃHCl (23 mg, 0.12 mmol), HOBt (16.2 mg, 0.12 mmol) and
DIPEA (69.7 lL, 0.40 mmol) were sequentially added to a stirred
solution of 6-heptynoic acid (15.2 lL, 0.12 mmol) in CH2Cl2
(2 mL) at room temperature. The reaction mixture was left stirring
for 15 min and then added to a stirred solution of 4h (60.6 mg,
0.10 mmol) in dry CH2Cl2 (2 mL). The reaction mixture was stirred
at room temperature overnight and then, after reaction completion,
the solvent was removed under reduced pressure. The crude prod-
uct was diluted with EtOAc (20 mL) and washed with a saturated
solution of ammonium chloride (3 ꢀ 20 mL), saturated solution of
sodium bicarbonate (3 ꢀ 20 mL) and brine (1 ꢀ 20 mL). The organic
layer was dried over Na2SO4, and then the solvent removed under
reduced pressure. The residue was purified by Biotage™ flash chro-
matography, eluant conditions: from 50% of EtOAc and 50% of
Petroleum ether to 100% of EtOAc. Yield 92% (65 mg, MW 713.42,
[M+Na]+, 571.1 [M+2H]2+
.
4.5. Head–head dimer 8d—Scheme 3
4.5.1. Compound 12d
EDCꢃHCl (23 mg, 0.12 mmol), HOBt (16.2 mg, 0.12 mmol) and
DIPEA (69.7 lL, 0.40 mmol) were sequentially added to a stirred
solution of 1,10-decanedioic acid (9.7 mg, 0.048 mmol) in dry
CH2Cl2 (2 mL) at room temperature. The reaction mixture was left
stirring for 15 min and then added to a stirred solution of 4h
(60.6 mg, 0.10 mmol) in dry CH2Cl2 (2 mL). The reaction mixture
was stirred at room temperature overnight and then, after reaction
completion, the solvent was removed under reduced pressure. The
crude product was diluted with EtOAc (20 mL) and washed with a
saturated solution of ammonium chloride (3 ꢀ 20 mL), saturated
solution of sodium bicarbonate (3 ꢀ 20 mL) and brine (1 ꢀ 20
mL). The organic layer was dried over Na2SO4, and then the solvent
removed under reduced pressure. The residue was purified by Bio-
tage™ flash chromatography, eluant conditions: 1% of MeOH and
99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 68%
(48 mg, MW 1376.71, 0.034 mmol) of pure 12d. Analytical charac-
0.092 mmol) of pure 11c. Analytical characterization: ½a 2D0
ꢂ75 (c
ꢁ
0.90, CHCl3); 1H NMR (400 MHz, CDCl3): d: 7.92 (bs, 1H), 7.40–
7.15 (m, 11H), 6.23 (d, J = 8.4 Hz, 1H), 4.75 (d, J = 7.6 Hz, 1H),
4.46 (m, 2H), 3.77 (m, 1H), 3.60 (m, 1H), 2.88 (s, 6H), 2.85 (m,
1H), 2.47 (m, 1H), 2.23 (m, 4H), 2.00–1.55 (m, 14H), 1.50 (s, 9H),
1.45–1.05 (m, 2H), 0.94 (m, 1H); 13C NMR (100 MHz, CDCl3): d:
173.1, 172.9, 171.7, 169.3, 142.1, 128.5, 127.5, 127.4, 127.3,
127.2, 68.4, 61.1, 58.9, 56.8, 53.8, 40.0, 36.2, 34.5, 33.2, 28.4,
28.1, 25.5, 24.8, 21.6, 18.2, 10.8. ESI-MS: m/z 714.2 [M+H]+.
4.7.2. Compound 12g
A stirred suspension of compound 11c (71.4 mg, 0.10 mmol)
and Cu(OAc)2 (127.1 mg, 0.70 mmol) in acetonitrile (20 mL) was
refluxed for 30 min. After reaction completion, the solvent was re-
moved under reduced pressure. The crude product was diluted
with EtOAc (20 mL) and washed with a saturated solution of
amonium chloride (3 ꢀ 10 mL) and brine (1 ꢀ 10 mL). Finally, the
crude product was purified by Biotage™ flash chromatography,
eluant conditions: 1% of MeOH and 99% of CH2Cl2 to 10% of MeOH
and 90% of CH2Cl2. Yield 84% (60 mg, MW 1425.83, 0.042 mmol) of
terization: ½a D20
ꢁ
ꢂ96 (c 1.34, CHCl3); 1H NMR (400 MHz, CDCl3):
d: 7.82 (bs, 2H), 7.30–7.05 (m, 22H), 6.07 (m, 2H), 6.14 (d,
J = 8.0 Hz, 2H), 4.66 (d, J = 7.2 Hz, 2H), 4,38 (m, 4H), 3.68 (m, 2H),
3.54 (m, 2H), 2.77 (m, 8H), 2.35 (m, 2H), 2.15 (m, 2H), 2.10 (m,
4H), 1.95–1.45 (m, 22H), 1.40 (s, 18H), 1.30–1.15 (m, 10H), 1.05
(m, 2H), 0.85 (m, 6H); 13C NMR (100 MHz, CDCl3): d: 173.6,
172.8, 171.8, 169.3, 142.2, 128.6, 127.4, 127.3, 127.2, 61.1, 60.5,
58.9, 56.8, 53.7, 40.0, 37.0, 34.5, 33.2, 29.4, 28.3, 25.8, 25.5, 21.5,
10.7. ESI-MS: m/z 1377.4 [M+H]+, 589.2 [M+2H]2+
.
pure 12g. Analytical characterization: ½a 2D0
ꢁ
ꢂ85 (c 1.1, CHCl3); 1H
NMR (400 MHz, CDCl3): d: 7.83 (bs, 2H), 7.35–7.05 (m, 22H), 6.80
(m, 2H), 6.14 (m, 2H), 4.65 (bs, 2H), 4.37 (m, 4H), 3.68 (m, 2H),
3.52 (m, 2H), 2.78 (s, 6H), 2.66 (m, 2H), 2.36 (m, 2H), 2.25–2.10
(m, 10H), 1.95–1.45 (m, 22H), 1.40 (s, 18H), 1.35–1.00 (m, 4H),
0.87 (bs, 6H); 13C NMR (100 MHz, CDCl3): d: 172.9, 171.7, 169.3,
142.1, 128.6, 127.5, 127.4, 127.3, 65.6, 61.1, 60.6, 58.9, 56.8, 53.7,
41.1, 40.0, 36.2, 34.5, 33.2, 31.8, 28.3, 28.0, 25.5, 24.9, 21.6, 19.0,
4.5.2. Compound 8d
TFA (450 lL, 5.0 mmol) was added to a stirred solution of com-
pound 12d (37.8 mg, 0.028 mmol) in CH2Cl2 (2 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from 70% of H2O (0.2% TFA) and 30% of
CH3CN (0.2% TFA) to 30% of H2O (0.2% TFA) and 70% of CH3CN
(0.2% TFA), flow rate 12 mL/min, 20 min runs. Yield 64% (25.3 mg,
MW 1405.59, 0.018 mmol) of pure 8d. Analytical characterization:
10.8. ESI-MS: m/z 1426.6 [M+H]+, 613.2 [M+2H]2+
.
4.7.3. Compound 8g
TFA (450 lL, 5.0 mmol) was added to a stirred solution of com-
½
a D20
ꢁ
ꢂ61 (c 1.06, CH3OH); 1H NMR (400 MHz, D2O): d: 8.80 (m, 2H),
pound 12g (94.1 mg, 0.066 mmol) in CH2Cl2 (2 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from 70% of H2O (0.2% TFA) and 30% of
CH3CN (0.2% TFA) to 30% of H2O (0.2% TFA) and 70% of CH3CN
(0.2% TFA), flow rate 12 mL/min, 20 min runs. Yield 41% (39 mg,
7.40–7.20 (m, 20H), 6.14 (s, 2H), 4.67 (m, 4H), 4.00 (m, 2H), 3.87
(bs, 2H), 3.44 (d, J = 13.6 Hz, 2H), 3.01 (t, J = 10 Hz, 2H), 2.70 (s,
6H), 2.85–2.70 (m, 6H), 2.70–1.75 (m, 16H), 1.70–1.55 (m, 8H),
1.32 (s, 8H), 1.05 (d, J = 7.0 Hz, 6H); 13C NMR (100 MHz, D2O): d:
175.1, 171.6, 169.6, 167.5, 141.7, 141.5, 128.3, 128.1, 127.2,
127.1, 126.9, 62.6, 61.5, 58.2, 57.0, 54.3, 40.9, 38.2, 35.7, 32.7,

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6701
MW 1453.64, 0.027 mmol) of pure 8g. Analytical characterization:
solution of Cbz-b-Ala-OH (26.8 mg, 0.12 mmol) in CH2Cl2 (2 mL)
at room temperature. The reaction mixture was left stirring for
15 min and then added to a stirred solution of 4h (60.6 mg,
0.10 mmol) in dry CH2Cl2 (2 mL). The reaction mixture was stirred
at room temperature overnight and then, after reaction comple-
tion, the solvent was removed under reduced pressure. The crude
product was diluted with EtOAc (20 mL) and washed with a satu-
rated solution of ammonium chloride (3 ꢀ 20 mL), saturated solu-
tion of sodium bicarbonate (3 ꢀ 20 mL) and brine (1 ꢀ 20 mL). The
organic layer was dried over Na2SO4, and then the solvent removed
under reduced pressure. The residue was purified by Biotage™
flash chromatography, eluant conditions: from 50% of EtOAc and
50% of Petroleum ether to 100% of EtOAc. Yield 99% (80 mg, MW
811.00, 0.099 mmol) of pure 11d. Analytical characterization:
½
a D20
ꢁ
ꢂ84 (c 1.16, CH3OH); 1H NMR (400 MHz, D2O): d: 8.80 (m,
2H), 7.40–7.20 (m, 20H), 6.14 (s, 2H), 4.67 (m, 4H), 4.02 (bs, 2H),
3.87 (bs, 2H), 3.44 (d, J = 13.2 Hz, 2H), 3.07 (t, J = 11.2 Hz, 2H),
2.72 (s, 6H), 2.40–2.20 (m, 10H), 2.20–180 (m, 16H), 1.75–1.60
(m, 8H), 1.60–1.45 (m, 4H), 1.05 (d, J = 7.1 Hz, 6H); 13C NMR
(100 MHz, D2O): d: 174.6, 171.6, 169.6, 167.5, 141.7, 141.5,
128.3, 128.1, 127.3, 127.2, 127.0, 126.9, 76.3, 65.4, 62.6, 61.6,
58.3, 57.0, 54.3, 40.9, 38.3, 35.1, 32.7, 31.7, 31.0, 30.2, 27.7, 27.5,
24.8, 23.2, 18.1, 7.8. ESI-MS: m/z 1226.0 [M+H]+, 613.7 [M+2H]2+
4.8. Head–head dimer 8h—Scheme 5
4.8.1. Compound 12h
.
A
0.9 M aqueous solution of sodium ascorbate (61
l
l
L,
L,
½
a D20
ꢁ
ꢂ118 (c 1.14, CHCl3); 1H NMR (400 MHz, CDCl3): d: 7.80 (d,
0.055 mmol) and a 0.3 M aqueous solution of Cu(OAc)2 (83
J = 8.0 Hz, 2H), 7.30–7.15 (m, 14H), 7.10 (d, J = 7.6 Hz, 2H), 6.13
(d, J = 8.8 Hz, 2H), 5.02 (t, J = 12.0 Hz, 2H), 4.63 (d, J = 7.6 Hz, 1H),
4.31 (m, 2H), 3.62 (m, 1H), 3.40 (m, 3H), 2.75 (m, 3H), 2.35 (m,
3H), 2.12 (m, 1H), 1.90–1.50 (m, 8H), 1.39 (s, 9H), 1.35 (m, 1H),
1.05 (m, 1H), 0.85 (t , J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d:.173.0, 171.6, 169.3, 156.4, 136.7, 128.7, 128.6, 128.4, 128.0,
127.4, 127.3, 127.2, 80.4, 66.5, 61.1, 60.8, 58.8, 56.8, 53.8, 41.3,
40.1, 37.3, 35.9, 34.5, 33.1, 32.0, 30.1, 28.4, 25.5, 21.8, 10.8. ESI-
MS: m/z 811.6 [M+H]+.
0.025 mmol) were sequentially added to a stirred solution of com-
pound 11c (0.10 mmol) and 1,4-(40-azidobutyl)benzene61 (13.6 mg,
0.05 mmol) in a 1:1 mixture of H2O/tBuOH (300
lL). The reaction
mixture was stirred overnight at room temperature and then the
solvent was removed under reduced pressure. The crude product
was dilutedwith EtOAc (20 mL)andwashedwith a saturatedsolution
of amonium chloride (3 ꢀ 20 mL) and brine (1 ꢀ 20 mL). The residue
was purified by Biotage™ flash chromatography, eluant conditions:
1% of MeOH and 99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2.
Yield 32% (27 mg, MW 1700.21, 0.016 mmol) of pure 12h. Analytical
4.9.2. Compound 12i
characterization: ½a 2D0
ꢁ
ꢂ84 (c 1.16, CHCl3); 1H NMR (400 MHz,
A solution of compound 11d (67.6 mg, 0.1 mmol) in EtOH/H2O
9:1 (10 mL) was Cbz-deprotected by continuous flow hydrogena-
tion using the H-Cube™ system. The hydrogenation was performed
using a Pd/C catalyst cartridge column (CatCart™). The reactor
pressure and temperature were fixed at 10 bar and 40 °C, and a
flow rate of 0.8 ml/min was set. After reaction completion, the sol-
vent was removed under reduced pressure.
CDCl3): d: 7.84 (bs, 2H), 7.37 (bs, 2H), 7.35–7.15 (m, 22H), 7.09
(d, J = 6.8 Hz, 2H), 6.98 (s, 4H), 6.13 (d, J = 7.6 Hz, 2H), 4.65 (d,
J = 6.8 Hz, 2H), 4.48 (m, 4H), 4.20 (m, 4H), 3.70 (m, 2H), 3.46 (m,
2H), 2.78 (s, 6H), 2.75 (m, 2H), 2.65 (s, 4H), 2.53 (t, J = 6.4 Hz,
4H), 2.34 (m, 2H), 2.17 (m, 6H), 1.95–1.50 (m, 28H), 1.40 (s,
18H), 1.39 (m, 2H), 1.05 (m, 2H), 0.85 (bs, 6H); 13C NMR
(100 MHz, CDCl3): d: 174.9, 173.0, 171.8, 169.4, 150.0, 140.5,
139.0, 1.38.0, 138.6, 128.4, 127.5, 127.3, 120.6, 80.5, 61.2, 60.6,
59.0, 56.9, 53.8, 50.0, 41.3, 40.1, 36.3, 34.8, 34.4, 33.2, 31.8, 30.5,
29.8, 28.9, 28.3, 25.6, 25.3, 21.7, 20.7, 10.8. ESI-MS: m/z 1699.6
The crude hydrogenation product was dissolved in EtOH (1 mL),
and then diethyl squarate (7.1 lL, 0.048 mmol), Et3N (55.8 lL,
0.4 mmol) and a catalytic amount of DMAP (1.2 mg, 0.01 mmol)
were sequentially added at room temperature. The reaction mix-
ture was left stirring overnight at room temperature. After reaction
completion, the solvent was removed under reduced pressure, the
crude product was diluted with EtOAc (20 mL) and washed with
water (3 ꢀ 20 mL). The organic layer was dried over Na2SO4, and
then the solvent was removed under reduced pressure. Finally,
the crude product was purified by Biotage™ flash chromatography,
eluant conditions: 1% of MeOH and 99% of CH2Cl2 to 10% of MeOH
and 90% of CH2Cl2. Yield 92% (63 mg, MW 1431.75, 0.044 mmol) of
[M+H]+, 850.8 [M+2H]2+
.
4.8.2. Compound 8h
TFA (450 lL, 5.0 mmol) was added to a stirred solution of com-
pound12h (21.2 mg, 0.013 mmol)inCH2Cl2 (2 mL).Thereaction mix-
ture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from70%of H2O (0.2% TFA)and30% ofCH3CN
(0.2% TFA) to 30% of H2O (0.2% TFA) and 70% of CH3CN (0.2% TFA), flow
rate 12 mL/min, 20 min runs. Yield 92% (24 mg, MW 1728.01,
pure 12i. Analytical characterization: ½a 2D0
ꢁ
ꢂ88 (c 1.60, CHCl3); 1H
NMR (400 MHz, CDCl3): d: 7.76 (d, J = 8.4 Hz, 2H), 7.30–7.10 (m,
22H), 6.13 (d, J = 8.8 Hz, 2H), 4.63 (d, J = 7.2 Hz, 2H), 4.35 (m, 4H),
3.75 (m, 5H), 3.25 (bs, 2H), 2.95 (bs, 2H), 2.73 (s, 6H), 2.42 (bs,
4H), 2.30 (m, 2H), 2.18 (m, 2H), 1.95–1.60 (m, 14H), 1.50 (m,
2H), 1.40 (s, 18H), 1.20 (m, 2H), 0.84 (t, J = 6.8 Hz, 6H); 13C NMR
(100 MHz, CDCl3): d: 171.4, 169.6, 142.1, 141.2, 128.7, 127.4,
127.2, 61.2, 58.8, 56.8, 53.8, 41.5, 40.8, 39.9, 36.9, 34.0, 33.1,
31.8, 30.7, 25.8, 21.6, 10.5. ESI-MS: m/z 1432.0 [M+H]+.
0.011 mmol) of pure 8h. Analytical characterization: ½a D20
ꢂ99 (c
ꢁ
1.16, CH3OH); 1H NMR (400 MHz, D2O): d: 8.80 (m, 2H), 7.76 (s,
2H), 7.40–7.15 (m, 20H), 7.06 (s, 4H), 6.14 (s, 2H), 4.66 (m, 4H),
4.37 (t, J = 6.4 Hz, 4H), 4.00 (m, 2H), 3.87 (m, 2H), 3.44 (d,
J = 13.2 Hz, 2H), 3.05 (t, J = 11.6 Hz, 2H), 2–74–2.68 (m, 10H),
2.59 (t, J = 6.8 Hz, 4H), 2.26 (m, 6H), 2.15–1.75 (m, 21H), 1.70–
1.50 (m, 17H), 1.06 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, D2O):
d: 174.7, 171.6, 169.6, 167.5, 147.1, 141.7, 141.6, 139.2, 128.3,
128.1, 127.3, 127.1, 126.9, 122.2, 62.6, 61.6, 58.3, 57.1, 54.3, 50.0,
41.0, 38.3, 35.3, 34.2, 32.7, 31.7, 31.0, 30.2, 29.3, 28.5, 28.0, 27.6,
4.9.3. Compound 8i
TFA (450 lL, 5.0 mmol) was added to a stirred solution of com-
pound 12i (41.5 mg, 0.029 mmol) in CH2Cl2 (2 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from 70% of H2O (0.2% TFA) and 30% of
CH3CN (0.2% TFA) to 30% of H2O (0.2% TFA) and 70% of CH3CN
(0.2% TFA), flow rate 12 mL/min, 20 min runs. Yield 83% (35 mg,
MW 1459.56, 0.024 mmol) of pure 8i. Analytical characterization:
25.0, 24.3, 23.2, 7.8. ESI-MS: m/z 1500.2 [M+H]+, 751.2 [M+2H]2+
.
4.9. Head–head dimer 8i—Scheme 6
4.9.1. Compound 11d
EDCꢃHCl (23 mg, 0.12 mmol), HOBt (16.2 mg, 0.12 mmol) and
½ ꢁ
a D20
ꢂ113 (c 1.20, CH3OH); 1H NMR (400 MHz, D2O): d:
DIPEA (69.7 L, 0.40 mmol) were sequentially added to a stirred
l

6702
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
7.35–7.25 (m, 20H), 6.00 (s, 2H), 4.57 (d, J = 10.0 Hz, 2H), 4.48 (dd,
J = 7.6, 5.2 Hz, 2H), 3.95 (m, 2H), 3.88 (dd, J = 7.2, 5.2 Hz, 2H), 3.71
(m, 4H), 3.30 (dd, J = 13.6, 3.6 Hz, 2H), 2.99 (dd, J = 13.6, 9.6 Hz,
2H), 2.64 (s, 6H), 2.44 (m, 4H), 2.20–2.05 (m, 4H), 1.95–1.85 (m,
8H), 1.80–1.60 (m, 6H), 1.50–1.35 (m, 4H), 0.94 (t, J = 7.2 Hz, 6H);
13C NMR (100 MHz, D2O): d: 181.7, 172.9, 172.0, 169.9, 168.4,
167.9, 141.3, 141.0, 128.8, 127.6, 127.5, 127.3, 127.0, 62.7, 61.6,
58.2, 57.4, 54.5, 40.7, 37.5, 37.0, 32.4, 31.6, 30.7, 27.7, 23.4, 8.3.
4.10.3. Compound 9a
TFA (450 L, 5.0 mmol) was added to a stirred solution of com-
l
pound 14a (30.4 mg, 0.022 mmol) in CH2Cl2 (2 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from 90% of H2O (0.2% TFA) and 10% of
MeOH/iPrOH 6:4 (0.2% TFA) to 100% of MeOH/iPrOH 6:4 (0.2%
TFA), flow rate 12 ml/min, 20 min runs. Yield 59% (18 mg, MW
1409.54, 0.013 mmol) of pure 9a. Analytical characterization:
ESI-MS: m/z 1231.9 [M+H]+, 616.5 [M+2H]2+
.
½
a D20
ꢁ
ꢂ110 (c 0.41, CH3OH); 1H NMR (400 MHz, D2O): d: 7.53 (s,
4.10. Tail–tail dimer 9a—Scheme 7
4.10.1. Compound 13a
2H), 7.20–7.05 (m, 10H), 6.59 (s, 4H), 6.10 (s, 2H), 4.57 (d,
J = 9.2 Hz, 2H), 4.38 (bs, 2H), 3.96 (bs, 4H), 3.81 (m, 4H), 3–49
(bs, 4H), 2.62 (s, 6H), 2.08 (bs, 4H), 2.00–1.80 (m, 10H), 1.75–
1.55 (m, 8H), 1.50–1.35 (m, 8H), 1.08 (bs, 4H), 0.88 (d, J = 7.2 Hz,
6H); 13C NMR (100 MHz, D2O): d: 172.3, 170.7, 168.0, 147.9,
139.4, 139.1, 128.8, 128.2, 128.0, 127.0, 123.0, 63.1, 62.7, 61.6,
58.5, 54.3, 50.0, 49.9, 39.0, 32.3, 31.5, 29.4, 28.9, 27.7, 27.5, 23.4,
EDCꢃHCl (69 mg, 0.36 mmol), HOBt (48.6 mg, 0.36 mmol) and DI-
PEA (209 L, 1.20 mmol) were sequentially added to a stirred solu-
l
tion of 4d (132.5 mg, 0.3 mmol) in CH2Cl2 (10 mL) at room
temperature. The reaction mixture was left stirring for 15 min and
then added to a stirred solution of (S)-3-amino-3-phenylprop-1-
yne (13.1 mg, 0.10 mmol) in dry CH2Cl2 (10 mL). The reaction
mixture was stirred at room temperature overnight and then, after
reaction completion, the solvent was removed under reduced pres-
sure. The crude product was diluted with EtOAc (20 mL) and washed
with a saturated solution of ammonium chloride (3 ꢀ 20 mL), satu-
rated solution of sodium bicarbonate (3 ꢀ 20 mL) and brine
(1 ꢀ 20 mL). The organic layer was dried over Na2SO4, and then
the solvent was removed under reduced pressure. The residue was
purified by Biotage™ flash chromatography, eluant conditions:
50% of EtOAc and 50% of Petroleum ether to 100% of EtOAc. Yield
67% (112 mg, MW 554.69, 0.201 mmol) of pure 13a. Analytical
8.3. ESI-MS: m/z 1181.9 [M+H]+, 1204.2 [M+Na]+, 591.8 [M+2H]2+
4.11. Tail–tail dimer 9b—Schemes 8 and 9
4.11.1. Compound 16a
.
HOBt (104 mg, 0.77 mmol), HBTU (292 mg, 0.77 mmol) and
Sym-collidine (169.1 L, 1.28 mmol) were sequentially added to
stirred solution of commercially available 15a (46.2 mg,
l
a
0.32 mmol) and N-Boc-(S)-2-Phenylglycine (200.9 mg, 0.80 mmol)
in dry DMF (2 mL) at 0 °C. The reaction mixture was left stirring
for 2 h and then, after reaction completion, the mixture was di-
luted with EtOAc (20 mL) and washed with a saturated solution
of ammonium chloride (3 ꢀ 20 mL), saturated solution of sodium
bicarbonate (3 ꢀ 20 mL) and brine (1 ꢀ 20 mL. The organic layers
were dried over Na2SO4, and then the solvent was removed under
reduced pressure. The residue was purified by Biotage™ flash chro-
matography, eluant conditions: from 1% of MeOH and 99% of
CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 94% (183 mg,
MW 610.80, 0.30 mmol) of pure 16a. Analytical characterization:
1H NMR (400 MHz, CDCl3):d: 7.38–7.25 (m, 10H), 5.76 (m, 2H),
5.25 (m, 2H), 3.32 (bs, 1H), 3.23 (m, 2H), 3.13 (bs, 1H), 1.57 (s,
4H), 1.42 (m, 18H), 1.21 (d, J = 11.2 Hz, 6H); 13C NMR (100 MHz,
CDCl3): d: 129.0, 128.9, 128.3, 127.2, 39.7, 39.7, 29.2, 28.8, 28.3,
26.4. ESI-MS: m/z 611.4 [M+H]+.
characterization: ½a 2D0
ꢁ
ꢂ116 (c 1.5, CHCl3); 1H NMR (400 MHz,
CDCl3): d: 7.65 (d, J = 7.6 Hz, 1H), 7.36 (m, 3H), 7.20 (m, 3H), 5.87
(d, J = 6.8 Hz, 1H), 4.61 (d, J = 6.0 Hz, 1H), 4.42–4.30 (m, 2H), 3.67
(m, 1H), 3.47 (d, J = 11.6 Hz, 1H), 3.14 (d, J = 12.0, 1H), 2.78 (s,
3H), 2.42 (s, 1H), 2.36 (m, 1H), 2.18 (m, 1H), 2.00–1.70 (m, 4H),
1.70–1.55 (m, 3H), 1.44 (s, 9H), 0.97 (m, 1H), 0.84 (m, 4H); 13C
NMR (100 MHz, CDCl3): d: 171.7, 138.9, 128.9, 126.8, 73.0, 64.1,
60.8, 60.2, 58.7, 53.6, 44.4, 41.5, 34.6, 33.2, 31.0, 30.0, 28.4, 25.4,
21.4, 10.6. ESI-MS: m/z 555.4 [M+H]+, 577.3 [M+Na]+.
4.10.2. Compound 14a
A 0.9 M water solution of sodium ascorbate (61
and a 0.3 M water solution of Cu(OAc)2 (83 L, 0.025 mmol) were
sequentially added to a stirred solution of compound 13a (61 mg,
0.11 mmol) and (13.6 mg,
1,4-(40-azidobutyl)benzene61
0.05 mmol) in a 1:1 mixture of H2O/tBuOH (300
L). The reaction
lL, 0.055 mmol)
l
4.11.2. Compound 17a
TFA (450 lL, 5.0 mmol) was added to a stirred solution of com-
pound 16a (165 mg, 0.27 mmol) in CH2Cl2 (3 mL). The reaction
mixture was left stirring at room temperature and then concen-
trated under reduced pressure to give a crude residue which was
used without purification. Quantitative yield (172.4 mg, MW
638.56, 0.27 mmol) of 17a.
l
mixture was stirred overnight at room temperature and then the
solvent was removed under reduced pressure. The crude product
was diluted with EtOAc (20 mL) and washed with a saturated solu-
tion of ammonium chloride (3 ꢀ 20 mL) and brine (1 ꢀ 20 mL). The
organic layer was dried over Na2SO4, and then the solvent was re-
moved under reduced pressure. Finally, the residue was purified by
Biotage™ flash chromatography, eluant conditions: 1% of MeOH
and 99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 74%
(51 mg, MW 1381.74, 0.037 mmol) of pure 14a. Analytical charac-
4.11.3. Compound 14b
HOBt (32.4 mg, 0.24 mmol), HBTU (91 mg, 0.24 mmol) and
Sym-collidine (52.9 lL, 0.4 mmol) were sequentially added to a
stirred solution of 4d (110.4 mg, 0.25 mmol) and 17a (63.9 mg,
0.10 mmol) in dry DMF (2 mL) at 0 °C. The reaction mixture was
left stirring for 2 h and then, after reaction completion, the mixture
was diluted with EtOAc (20 mL) and washed with a saturated solu-
tion of ammonium chloride (3 ꢀ 20 mL), saturated solution of so-
dium bicarbonate (3 ꢀ 20 mL) and brine (1 ꢀ 20 mL). The organic
layers were dried over Na2SO4, and then the solvent was removed
under reduced pressure. The residue was purified by Biotage™
flash chromatography, eluant conditions: 1% of MeOH and 99% of
CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 97% (122 mg,
MW 1257.59, 0.094 mmol) of pure 14b. Analytical characteriza-
terization: ½a D20
ꢁ
ꢂ83 (c 1.15, CHCl3); 1H NMR (400 MHz, CDCl3): d:
7.94 (m, 2H), 7.42 (m, 2H), 7.30–7.10 (m, 12H), 6.96 (s, 4H), 6.20 (d,
J = 6.4 Hz, 2H), 4.64 (bs, 2H), 4.38 (m, 4H), 4.23 (bs, 4H), 3.70 (m,
2H), 3.53 (d, J = 12.0 Hz, 2H), 3.21 (d, J = 12.0 Hz, 2H), 2.79 (s,
6H), 2.53 (bs, 4H), 2.27 (m, 2H), 2.16 (m, 2H), 2.05–1.60 (m,
18H), 1.56 (m, 4H), 1.42 (s, 18H), 1.14 (m, 4H), 0.84 (bs, 6H); 13C
NMR (100 MHz, CDCl3): d: 173.0, 171.5, 169.6, 140.9, 139.0,
128.6, 128.4, 127.7, 127.1, 121.4, 80.0, 64.3, 61.2, 60.2, 58.8, 53.8,
50.2, 50.0, 41.6, 34.7, 34.5, 33.1, 31.1, 29.7, 28.4, 28.2, 26.0, 21.5,
10.7. ESI-MS: m/z 1382.1 [M+H]+, 691.7 [M+2H]2+
.

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6703
tion: ½a D20
ꢁ
ꢂ118 (c 1.23, CHCl3); 1H NMR (400 MHz, CDCl3): d: 8.43
20H), 1.22 (m, 2H), 0.85 (bs, 6H) ; 13C NMR (100 MHz, CDCl3): d:
171.5, 171.0, 169.6, 140.7, 139.0, 128.7, 128.4, 127.9, 127.1,
121.6, 61.0, 59.0, 58.7, 53.6, 53.2, 50.3, 50.0, 34.7, 34.1, 33.3, 32.1,
29.7, 28.4, 28.2, 26.0, 21.4, 10.6. ESI-MS: m/z 1433.1 [M+H]+,
(d, J = 8 Hz, 2H), 8.17 (t, J = 5.6 Hz, 2H), 7.86 (bs, 2H), 7.40–7.25 (m,
10H), 5.36 (d, J = 8 Hz, 2H), 4.61 (dd, J = 7.2, 4 Hz, 2H), 4.48–4.36
(m, 4H), 3.89 (m, 2H), 3.45 (bs, 2H), 3.25 (m, 2H), 3.00 (m, 4H),
2.70 (s, 6H), 2.16 (m, 2H), 2.14 (m, 2H), 2.00–1.75 (m, 8H), 1.70–
1.45 (m, 10H), 1.40 (s, 18H), 1.35 (m, 4H), 1.15 (s, 8H), 0.80 (bs,
6H); 13C NMR (100 MHz, CDCl3): d: 171.2, 170.3, 169.9, 139.4,
128.6, 127.8, 127.5, 79.5, 63.1, 61.1, 58.0, 56.8, 53.9, 41.0, 39.0,
33.2, 32.8, 30.2, 29.3, 29.0, 28.5, 28.1, 26.6, 11.2 . ESI-MS: m/z
1257.9 [M+H]+.
666.7 [M+2H]2+
.
4.13.2. Compound 14g
A 1 N solution of (CH3)3P in toluene (105 lL, 0.105 mmol) was
added dropwise to a stirred solution of compound 14f (50.1 mg,
0.035 mmol) in dry CH2Cl2 (10 mL) under argon at room tempera-
ture. After 2 h, a 1 N HCl aqueous solution (10 mL) was added to
the reaction mixture, and stirring at room temperature was contin-
ued for further 20 min. After reaction completion, the reaction
mixture was neutralized with sodium bicarbonate and extracted
with CH2Cl2 (3 ꢀ 20 mL). The organic layers were combined and
dried over Na2SO4, and the solvent was removed under reduced
pressure. The crude product did not require any further purifica-
tion, and was characterized as such. Yield 99% (48 mg, MW
1379.77, 0.035 mmol) of pure 14g. Analytical characterization:
4.11.4. Compound 9b
TFA (450 lL, 5.0 mmol) was added to a stirred solution of com-
pound 14b (93.1 mg, 0.074 mmol) in CH2Cl2 (2 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from 70% of H2O (0.2% TFA) and 30% of
CH3CN (0.2% TFA) to 30% of H2O (0.2% TFA) and 70% of CH3CN
(0.2% TFA), flow rate 12 ml/min, 20 min runs. Yield 58% (55 mg,
MW 1285.35, 0.043 mmol) of pure 9b. Analytical characterization:
½
a D20
ꢁ
ꢂ120 (c 0.48, CHCl3); 1H NMR (400 MHz, CDCl3): d: 8.35 (m,
4H), 7.88 (m, 2H), 7.35–7.15 (m, 12H), 6.93 (m, 4H), 6.14 (d,
J = 6.4 Hz, 2H), 4.62 (bs, 2H), 4.40 (m, 4H), 4.24 (bs, 4H), 3.70 (m,
2H), 3.00–2.65 (m, 10H), 2.52 (bs, 4H), 2.30–2.05 (m, 4H), 2.00–
1.55 (m, 20H), 1.54 (m, 4H), 1.39 (s, 18H), 1.19 (m, 4H), 0.84 (bs,
6H); 13C NMR (100 MHz, CDCl3): d: 173.2, 171.7, 169.2, 141.9,
139.3, 129.6, 128.2, 127.5, 127.4, 122.4, 81.0, 64.5, 61.4, 60.1,
58.8, 53.8, 50.2, 50.0, 41.6, 35.7, 33.5, 33.1, 31.5, 29.7, 28.4, 28.2,
½
a D20
ꢁ
ꢂ94 (c 1.44, CH3OH); 1H NMR (400 MHz, D2O): d: 7.41 (m,
10H), 5.29 (s, 2H), 4.70 (m, 2H), 4.53 (dd, J = 8.0, 4.4 Hz, 2H), 4.05
(m, 2H), 3.88 (dd, J = 7.2, 5.2 Hz, 2H), 3.61 (m, 2H), 3.25 (m, 2H),
3.05 (m, 2H), 2.68 (s, 6H), 2.25 (m, 2H), 2.15 (m, 2H), 2.05 (m,
4H), 1.95 (m, 6H), 1.85 (m, 3H), 1.75 (m, 4H), 1.60 (m, 2H), 1.35
(m, 4H), 1.012(bs, 8H), 0.96 (t, J = 8.0 Hz, 6H); 13C NMR
(100 MHz, D2O): d: 173.2, 171.6, 171.2, 168.3, 136.1, 129.2, 129.0,
127.3, 63.2, 62.8, 61.8, 58.8, 58.2, 54.5, 39.3, 39.0, 32.4, 31.5,
31.4, 29.5, 28.1, 27.8, 25.6, 23.4, 8.2. ESI-MS: m/z 1057.9 [M+H]+.
26.0, 21.5, 10.4. ESI-MS: m/z 1380.1 [M+H]+, 691.2 [M+2H]2+
.
4.13.3. Compound 14h
NaBH(OAc)3 (16.5 mg, 0.078 mmol) was added to a stirred solu-
4.12. Tail–tail dimers 9c–e—Schemes 8 and 9
tion of compound 14g (41.4 mg, 0.03 mmol) and benzaldehyde
(5.5 lL, 0.054 mmol) in dry CH2Cl2 (5 mL). The reaction mixture
The tail–tail dimers 9c, 9d and 9e were prepared similarly to 9b.
The detailed synthetic protocols leading to their synthesis, and
their full synthetic characterization are reported in the Supplemen-
tary data.
was stirred overnight at room temperature, and then the solvent
was removed under reduced pressure. Finally, the crude product
was purified by Biotage™ flash chromatography, eluant condi-
tions: 1% of MeOH and 99% of CH2Cl2 to 10% of MeOH and 90% of
CH2Cl2. Yield 45% (21 mg, MW 1560.32, 0.0135 mmol) of pure
4.13. Tail–tail dimer 9f—Scheme 10
14h. Analytical characterization: ½a 2D0
ꢁ
ꢂ150 (c 0.70, CHCl3); 1H
NMR (400 MHz, CDCl3): d: 8.25 (bs, 2H), 8.03 (m, 2H), 7.40–7.24
(m, 20H), 7.18 (m, 2H), 7.05 (s, 4H), 6.28 (d, J = 7.6 Hz, 2H), 4,73
(bs, 2H), 4.49 (m, 4H), 4.30 (s, 4H), 3.95–3.75 (m, 6H), 2.95–2.75
(m, 6H), 2.70–2.50 (m, 8H), 2.35 (m, 2H), 2.23 (m, 2H), 2.25–1.55
(m, 22H), 1.49 (m, 20H), 1.24 (m, 2H), 0.93 (bs, 6H); 13C NMR
(100 MHz, CDCl3): d: 169.9, 147.8, 141.0, 139.1, 128.8, 128.6,
127.6, 127.2, 127.0, 121.5, 61.0, 58.4, 50.2, 50.0, 38.1, 34.7, 33.3,
33.2, 29.7, 28.5, 28.2, 26.0, 22.1, 10.6. ESI-MS: m/z 1561.4
4.13.1. Compound 14f
Dry TEA (83.6
lL, 0.6 mmol) and MsCl (46.4
lL, 0.6 mmol) were
stirred solution of compound 14a
sequentially added to
a
(0.1 mmol) in dry CH2Cl2 (4 mL) under argon at 0 °C. The reaction
mixture was stirred at room temperature for 2 h. After reaction
completion, the resulting mixture was diluted with CH2Cl2
(20 mL) and washed with a saturated solution of ammonium chlo-
ride (3 ꢀ 20 mL). The combined organic layers were dried over
Na2SO4, and then the solvent was removed under reduced pressure.
The crude mesylate was dissolved in dry DMF (10 mL) under ar-
gon at room temperature, and then NaN3 (130 mg, 2 mmol) was
added. The reaction mixture was heated at 100 °C for 30 min, while
being irradiated in a microwave reactor. After reaction completion
most of the DMF was removed under reduced pressure, the crude
was diluted with EtOAc (20 mL) and washed with water
(3 ꢀ 20 mL). The organic layer was dried over Na2SO4, and then
the solvent was removed under reduced pressure. Finally, the
crude product was purified by Biotage™ flash chromatography.
eluant conditions: 1% of MeOH and 99% of CH2Cl2 to 10% of MeOH
and 90% of CH2Cl2. Yield 58% (83 mg, MW 1431.77, 0.058 mmol) of
[M+H]+, 781.0 [M+2H]2+
.
4.13.4. Compound 9f
TFA (90 lL, 1.0 mmol) was added to a stirred solution of com-
pound 14h (20.3 mg, 0.013 mmol) in CH2Cl2 (2 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from 90% of H2O (0.2% TFA) and 10% of
MeOH/iPrOH 6:4 (0.2% TFA) to 100% of MeOH/iPrOH 6:4 (0.2%
TFA), flow rate 12 ml/min, 20 min runs. Yield 92% (19 mg, MW
1587.83, 0.012 mmol) of pure 9f. Analytical characterization:
½
a D20
ꢁ
ꢂ121 (c 0.95, CH3OH); 1H NMR (400 MHz, D2O): d: 7.52 (s,
pure 14f. Analytical characterization: ½a 2D0
ꢁ
ꢂ105 (c 1.31, CHCl3); 1H
2H), 7.35 (s, 10H), 7.25–7.05 (m, 10H), 6.64 (s, 4H), 6.06 (s, 2H),
4.56 (d, J = 9.2 Hz, 2H), 4.37 (bs, 2H), 4.14 (s, 4H), 4.045 (bs, 4H),
3.89 (bs, 2H), 3.76 (bs, 2H), 3.03 (d, J = 12.0 Hz, 2H), 2.95 (t,
J = 11.2 Hz, 2H), 2.49 (s, 6H), 2.15 (bs, 4H), 2.12–1.90 (m, 8H),
1.90–1.70 (m, 8H), 1.70–1.65 (m, 2H), 1.50 (bs, 8H), 1.13 (bs, 4H),
0.84 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, D2O): d: 172.5, 168.9,
NMR (400 MHz, CDCl3): d: 8.03 (m, 2H), 7.40–7.20 (m, 12H), 7.05
(m, 6H), 6.29 (d, J = 7.6 Hz, 2H), 4.71 (d, J = 6.4 Hz, 2H), 4.58 (m,
2H), 4.49 (m, 2H), 4.33 (bs, 4H), 3.83 (m, 2H), 3.42 (bd,
J = 11.6 Hz, 2H), 3.16 (t, J = 12.8 Hz, 2H), 2.86 (s, 6H), 2.62 (m,
4H), 2.36 (m, 2H), 2.23 (m, 2H), 2.05–2.60 (m, 22H), 1.55 (bs,

6704
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
168.7, 147.5, 139.3, 139.1, 130.1, 129.9, 129.8, 129.3, 128.9, 128.2,
128.1, 127.0, 132.2, 62.5, 61.7, 58.0, 54.1, 51.4, 50.2, 50.1, 47.8,
35.2, 33.7, 32.2, 31.5, 29.7, 28.7, 28.4, 27.9, 27.4, 23.4, 8.1. ESI-
J = 7.6, 4.0, 1H), 4.56 (t, J = 9.6 Hz, 1H), 4.38 (bs, 1H), 3.97 (dd,
J = 14.8, 7.2 Hz, 1H), 3.78 (s, 3H), 2.87 (s, 3H), 2.65 (bd, 1H), 2.45–
2.28 (m, 3H), 2.20–1.70 (m, 9H), 1.50 (s, 9H), 0.92 (t, J = 7.6 Hz,
3H); 13C NMR (100 MHz, CDCl3): d: 172.3, 169.0, 60.4, 58.6, 54.4,
52.5, 37.5, 33.9, 33.4, 32.7, 28.4, 27.7, 21.6, 20.8, 10.7. ESI-MS:
m/z 465.5 [M+H]+.
MS: m/z 1360.0 [M+H]+, 680.6 [M+2H]2+
4.14. Tail–tail dimer 9g—Scheme 11
4.14.1. Compound 13b
.
4.14.3. Compound 13d
Dry TEA (223
l
L, 1.6 mmol) and Boc2O (349.2 mg, 1.6 mmol)
A solution of nitrile 13c (0.31 mmol) in EtOH (4 mL), and the
was flowed through a Raney Nickel catalyst cartridge (hydrogen
pressure 60 bar, temperature 60 °C, flow rate 0.5 mL/min) in a con-
tinuous flow hydrogenation H-Cube™ system. After reaction com-
pletion, the solvent was removed under reduced pressure and the
crude was used as such in the next reaction step.
were sequentially added to a stirred solution of compound 4c
(205 mg, 0.8 mmol) in dry CH2Cl2 (4 mL). The reaction mixture was
stirred at room temperature and monitored by LC–MS. After reaction
completion, the solution was diluted with CH2Cl2 (5 mL) and washed
oncewith5% citric acidand saturated NaHCO3. The organic layerwas
dried over Na2SO4 and evaporated under reduced pressure. The
crude product was used as such in the next reaction step.
Dry TEA (86.4 lL, 0.62 mmol) and Boc2O (135.3 mg, 0.62 mmol)
were sequentially added to a stirred solution of the crude amine in
dry CH2Cl2 (3 mL). The reaction mixture was stirred at room tem-
perature and monitored by LC–MS. After reaction completion, the
solution was diluted with CH2Cl2 (5 mL) and washed once with
5% citric acid and saturated NaHCO3. The organic layer was dried
over Na2SO4 and evaporated under reduced pressure. The crude
product was purified by flash chromatography, eluant conditions:
from 10% of EtOAc and 90% of petroleum ether to 100% of EtOAc.
Yield 44% (78 mg, MW 568.60, 0.137 mmol) of pure 13d. Analytical
characterization: 1H NMR (400 MHz, CDCl3): d: 6.88 (d, J = 4.8 Hz,
1H), 4.57 (m, 2H), 4.41 (dd, J = 9.2, 6.0 Hz, 1H), 4.94 (bd,
J = 8.4 Hz, 1H), 3.75 (s, 3H), 3.06 (bs, 2H), 2.84 (s, 3H), 2.25 (m,
1H), 2.20–2.03 (m, 3H), 2.0–1.85 (m, 2H), 1.80–1.55 (m, 6H), 1.49
(s, 9H), 1.43 (s, 9H), 1.28 (t, J = 7.2 Hz, 1H), 0.91 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, CDCl3): d: 172.5, 170.7, 155.9, 79.0, 60.2,
58.5, 55.0, 52.3, 38.4, 37.2, 33.5, 32.8, 32.3, 28.4, 27.8, 21.7, 21.0,
14.2, 10.7. ESI-MS: m/z 569.5 [M+H]+.
Dry TEA (335 lL, 2.4 mmol) and MsCl (92.9 lL, 1.2 mmol) were
sequentially added to a stirred solution of crude alcohol (270 mg,
0.78 mmol) in dry CH2Cl2 (4 mL) under argon at 0 °C. The reaction
mixture was stirred at room temperature overnight. After reaction
completion, the resulting mixture was diluted with CH2Cl2 (20 mL)
and washed with
a saturated ammonium chloride solution
(2 ꢀ 20 mL). The organic layer was dried over Na2SO4, and then
the solvent was removed under reduced pressure.
The crude product was dissolved in dry DMF (10 mL) and trea-
ted with Bu4N+CN- (322.2 mg, 1.2 mmol). The reaction mixture was
heated at 80 °C for 90 min in a microwave reactor. After reaction
completion most of the DMF was removed under reduced pressure,
the crude was diluted with EtOAc (20 mL) and washed with water
(3 ꢀ 20 mL). The organic layer was dried over Na2SO4, and then the
solvent was removed under reduced pressure. The crude product
was purified by Biotage™ flash chromatography, eluant condi-
tions: from 1% of MeOH and 99% of CH2Cl2 to 10% of MeOH and
90% of CH2Cl2. Yield 63% (245 mg, MW 365.0, 0.493 mmol) of pure
13b. Analytical characterization: 1H NMR (400 MHz, CDCl3): d:
5.83 (d, J = 6.8 Hz, 1H), 4.61 (dd, J = 8.0, 4.4 Hz, 1H), 4.25 (t,
J = 9.6 Hz, 1H); 3.92 (dd, J = 14.8, 7.6 Hz, 1H), 3.77 (s, 3H), 2.76
(dd, J = 15.2, 4.4 Hz, 1H), 2.42–2.25 (m, 3H), 2.15–1.65 (m, 7H),
1.45 (s, 9H); 13C NMR (100 MHz, CDCl3): d: 172.2, 169.5, 118.9,
80.2, 60.4, 58.7, 56.2, 52.4, 37.7, 34.0, 33.3, 32.8, 28.3, 27.7,20.9.
ESI-MS: m/z 753.4 [2 M+Na]+.
4.14.4. Compound 13e
A 2 N aqueous LiOH solution (0.5 mL) was slowly added to an
iced cooled, stirred solution of 13d (74 mg, 0.13 mmol) in 1,4-diox-
ane (1 mL). The reaction mixture was then stirred at room temper-
ature until complete hydrolysis of the starting material. After
reaction completion, the cloudy solution was concentrated, the
residue was taken up in CH2Cl2 (10 mL) and water (10 mL), and
acidified to pH ꢄ3 with 2 N aqueous HCl. The mixture was ex-
tracted with CH2Cl2 (2 ꢀ 10 mL), the combined organic layers were
dried over Na2SO4, and then the solvent was removed under re-
duced pressure. Crude 13e was used without purification in the
next reaction step.
4.14.2. Compound 13c
Compound 13b (0.49 mmol) was dissolved in 3 N methanolic
HCl solution (3 mL). The resulting mixture was stirred at room
temperature overnight, and then evaporated under reduced pres-
sure. The crude product was re-dissolved in CH2Cl2 (20 mL) and
washed once with a saturated solution of NaHCO3. The organic
layer was dried over Na2SO4, filtered and the solvent was removed
under reduced pressure. The crude product was used as such in the
next reaction step.
4.14.5. Compound 14i
HOBt (16.2 mg, 0.12 mmol), HBTU (45.5 mg, 0.12 mmol) and
Sym-collidine (26.4 lL, 0.2 mmol) were sequentially added to a
stirred solution of 17b (39.3 mg, 0.055 mmol) and crude 13e
(72.1 mg, 0.13 mmol) in dry DMF (2 mL) at 0 °C. The reaction mix-
ture was left stirring and monitored by LC–MS. After reaction com-
pletion, the mixture was diluted with EtOAc (20 mL) and
sequentially washed with 5% aqueous citric (20 mL) acid, saturated
aqueous sodium bicarbonate (20 mL) and brine (2 ꢀ 20 mL). The
organic layer was dried over Na2SO4, and then the solvent removed
under reduced pressure. The residue was purified by Biotage™
flash chromatography, eluant conditions: from 10% of EtOAc and
90% of petroleum ether to 100% of EtOAc. Yield 77% (66 mg, MW
1559.97, 0.042 mmol) of pure 7. Analytical characterization: 1H
NMR (400 MHz, CDCl3): d: 8.03 (d, J = 4.5 Hz, 2H), 7.32–7.02 (m,
10H), 7.01 (d, J = 8.4 Hz, 2H), 6.85 (bs, 2H), 5.32 (d, J = 4.5 Hz,
2H), 4.68 (d, J = 7.2 Hz, 2H), 4.48 (bs, 2H), 4.45 (dd, J = 9.6, 4.0 Hz,
2H), 3.86 (m, 2H), 3.62–3.25 (m, 16H), 3.10 (m, 4H), 2.86 (s, 6H),
2.32–2.15 (M, 4H), 2.05–1.60 (m, 24H), 1.52 (s, 18H), 1.43 (s,
18H), 1.28 (m, 2H), 0.93 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz,
Dry DIPEA (341.4 lL, 1.96 mmol) was added to a solution of N-
Boc, N-Me-(S)-ethylglycine (134.7 mg, 0.62 mmol), EDCꢃHCl
(115 mg, 0.6 mmol) and HOBt (73.3 mg, 0.6 mmol) in dry CH2Cl2
(3 mL) at room temperature under a nitrogen atmosphere. The
solution was stirred for 10 min before adding a solution of the
crude amine (180 mg, 0.49 mmol) in CH2Cl2 (2 mL). The reaction
mixture was stirred at room temperature and monitored by LC–
MS. After reaction completion, the solution was diluted with
CH2Cl2 (20 mL) and washed once with 5% citric acid and saturated
NaHCO3. The organic layer was dried over Na2SO4 and evaporated
under reduced pressure. The crude product was purified by flash
chromatography, eluant conditions: from 1% of MeOH and 99% of
CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 67% (152 mg,
MW 464.57, 0.327 mmol) of pure 13c. Analytical characterization:
1H NMR (400 MHz, CDCl3): d: 7.14 (d, J = 7.2 Hz, 1H); 4.62 (dd,

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6705
CDCl3): d: 171.9, 169.4, 156.0, 138.4, 128.9, 128.8, 128.2, 128.1,
127.2, 70.5, 70.0, 69.6, 61.2, 58.7, 57.3, 54.8, 38.1, 37.3, 34.5,
33.2, 28.5, 26.6, 10.8. ESI-MS: m/z 1559.9 [M+H]+.
characterization: 1H NMR (400 MHz, CDCl3): d: 7.93 (d, J = 6.8 Hz,
1H), 7.51 (d, J = 8.0 Hz, 1H), 7.36–7.27 (m, 5H), 6.37 (bs, 1H), 5.38
(d, J = 7 Hz, 1H), 4.71 (d, J = 6.8 Hz, 1H), 4.48 (m, 2H), 3.68 (m,
1H), 3.65 (m, 8H), 3.55 (m, 4H), 3.45 (m, 4H), 3.38 (m, 2H), 3.32
(m, 1H), 2.87 (s, 3H), 2.32 (m 1H), 2.22 (m, 1H), 2.10–1.40 (m,
3H), 1.35–1.70 (m, 5H), 1.52 (s, 9H), 1.30 (m, 2H), 0.93 (t,
J = 7 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 173.2, 172.4, 171.4,
170.0, 169.5, 138.2, 128.9, 128.2, 127.2, 70.7, 70.6, 70.5, 70.3,
70.0, 69.5, 64.3, 61.4, 61.0, 59.1, 58.8, 57.4, 53.9, 50.6, 41.6, 39.7,
34.4, 33.0, 32.8, 30.4, 28.4, 26.4, 21.6, 10.7 . ESI-MS: m/z 775.5
[M+H]+, 797.5 [M+Na]+.
4.14.6. Compound 9g
TFA (270 lL, 3.0 mmol) was added to a stirred solution of com-
pound 14i (61 mg, 0.04 mmol) in CH2Cl2 (5 mL). The reaction mix-
ture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified
by chromatography on a C18 reverse phase semi-preparative hplc
column, eluant conditions: from 70% of H2O (0.2% TFA) and 30%
of CH3CN (0.2% TFA) to 30% of H2O (0.2% TFA) and 70% of CH3CN
(0.2% TFA), flow rate 12 ml/min, 20 min runs. Yield 80% (52 mg,
MW 1615.57, 0.032 mmol) of pure 9g. Analytical characterization:
1H NMR (400 MHz, D2O): d: 7.47 (m, 10H), 5.28 (s, 2H), 4.60 (d,
J = 9.6 Hz, 2H), 4.49 (dd, J = 8.0, 5.2 hz, 2H), 4.05 (m, 2H), 3.50
(dd, J = 6.4, 3.2 Hz, 4H), 3.43 (dd, J = 5.2, 3.2 Hz, 4H), 3.32 (t,
J = 6.4 Hz, 4H), 3.29 (t, J = 6.8 Hz, 2H), 3.20–3.00 (m, 4H), 2.95 (m,
2H), 2,67 (s, 6H), 2.25 (m, 2H), 2.15 (m, 2H), 2.03 (m, 4H), 1.90
(m, 6H), 1.85–1.55 (m, 16H), 0.92 (t, J = 7.6, Hz, 6H); (100 MHz,
D2O): d: 173.3, 171.8, 170.4, 168.6, 135.9, 129.3, 127.3, 69.4, 69.3,
68.0, 62.6, 61.6, 58.4, 58.3, 56.1, 37.3, 36.4, 35.1, 32.4, 31.6, 30.5,
4.15.3. Compound 11e
DCC (22.7 mg, 0.11 mmol) and DMAP (2.4 mg, 0.02 mmol) were
sequentially added to a stirred solution of compound 4h (60 mg,
0.1 mmol) and 4-pentynoic acid (9.8 mg, 0.1 mmol) in dry CH2Cl2
(4 mL) at 0 °C. The reaction mixture was warmed to room temper-
ature over a period of 1 h. After reaction completion, the reaction
mixture was filtered, and the filtrate was washed with diethyl
ether. The organic phase was removed under reduced pressure,
and the crude product was purified by Biotage™ flash chromatog-
raphy, eluant conditions: from 1% of MeOH and 99% of CH2Cl2 to
10% of MeOH and 90% of CH2Cl2. Yield 93% (64 mg, MW 685.87,
29.8, 29.1, 28.1, 27.9, 23.5, 8.1. ESI-MS: m/z 580.6 [M+H]2+
1159.7 [M+H]+.
,
0.093 mmol) of pure 11e. Analytical characterization:
½ ꢁ
a D20
ꢂ116.0 ( c 0.90, CHCl3); 1H NMR (400 MHz, CDCl3): d: 7.90 (d,
J = 6.8 Hz, 1H), 7.28–7.09 (m, 11H), 6.13 (d, J = 8.0 Hz, 1H), 4.64
(d, J = 7.0 Hz, 1H), 4.45 (bs, 1H), 4.34 (m, 1H), 3.67 (m, 1H), 3.35
(m, 1H), 3.00–2.60 (m, 4H), 2.50–2.30 (m, 5H), 2.15 (m, 1H),
2.00–1.50 (m, 9H), 1.45 (m, 1H), 1.40 (s, 9H), 1.35–1.05 (m, 1H),
0.91 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 172.8,
171.7, 171.3, 169.3, 142.0, 141.2, 128.7, 128.6, 127.5, 127.4,
127.3, 127.2, 80.7, 69.0, 62.0, 58.9, 56.8, 54.0, 41.8, 39.9, 35.4,
34.5, 33.3, 33.0, 32.0, 28.3, 27.5, 25.5, 21.9, 15.1, 10.8. ESI-MS: m/
z 686.5 [M+H]+, 703.5 [M+Na]+.
4.15. Head–tail dimer 10a—Schemes 12 and 13
4.15.1. Compound 16e
HOBt (81 mg, 0.6 mmol), HBTU (227.5 mg, 0.6 mmol) and Sym-
collidine (132.1 L, 1 mmol) were sequentially added to a stirred
l
solution of commercially available 15e (110 mg, 0.503 mmol) and
N-Boc-(S)-2-Phenylglycine (158 mg, 0.63 mmol) in dry DMF
(3 mL) at 0 °C . The reaction mixture was left stirring and monitored
by LC–MS. After reaction completion, the mixture was diluted with
EtOAc (20 mL) and sequentially washed with 5% aqueous citric acid
(20 mL), saturated aqueous sodium bicarbonate (20 mL) and brine
(2 ꢀ 20 mL). The organic layer was dried over Na2SO4, and then
the solvent was removed under reduced pressure. The residue was
purified by Biotage™ flash chromatography, eluant conditions: from
1% of MeOH and 99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2.
Yield 74% (167 mg, MW 451.53, 0.37 mmol) of pure 16e. Analytical
characterization: 1H NMR (400 MHz, CDCl3): d: 7.32.7.20 (m, 5H),
6.28 (bs, 1H), 5.77 (bs, 1H), 5.07 (bs, 1H), 3.6–3.28 (m, 16H), 1.35
(s, 9H); 13C NMR (100 MHz, CDCl3): d: 129.0, 128.3, 127.2, 70.7,
70.6, 70.5, 70.3, 70.0, 69.6, 68.8, 58.5, 50.7, 39.6, 28.3. ESI-MS: m/z
452.5 [M+H]+, 474.5 [M+Na]+.
4.15.4. Compound 19a
A 0.9 M aqueous solution of sodium ascorbate (70
lL) and a
0.3 M aqueous solution of Cu(OAc)2 (50 L) were sequentially
l
added to a stirred solution of compound 18a (44 mg, 0.057 mmol)
and compound 11e (38 mg, 0.057 mmol) in a 1:1 mixture of
H2O/tBuOH (1 mL). The reaction mixture was stirred overnight at
room temperature and then the solvent was removed under re-
duced pressure. The residues were purified by Biotage™ flash chro-
matography, eluant conditions: from 1% of MeOH and 99% of
CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 61% (51 mg,
MW 1460.79, 0.035 mmol) of pure 19a. Analytical characteriza-
tion: 1H NMR (400 MHz, CDCl3): d: 7.95 (d, J = 6.8 Hz, 2H), 7.51
(m, 2H), 7.35–7.20 (m, 15H), 7.16 (d, J = 9.2 Hz, 2H), 6.75 (bs,
1H), 6.22 (d, J = 6.8 Hz, 1H), 5.43 (d, J = 7.2 Hz, 1H), 4.72 (dd,
J = 16, 7.6 Hz, 2H), 4.48 (m, 6H), 3.82 (m, 4H), 3.70–3.30 (m,
15H), 3.07 (t, J = 7.6 Hz, 2H), 2.85 (m, 6H), 2,.67 (m, 3H), 2.48 (m,
1H), 2.25 (m, 3H), 2.05 (m, 1H), 2.00–1.80 (m, 5H), 1.80–1.65 (m,
7H), 1.52 (s, 9H), 1.48 (s, 9H), 1.35 (m, 1H), 1.25 (m, 2H), 1.10
(m, 1H), 0.94 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3): d:
173.1, 171.4, 169.6, 169.4, 146.1, 142.1, 138.4, 128.8, 128.7,
128.6, 127.3, 127.2, 127.1, 123.0, 70.5, 70.4, 70.2, 69.5, 69.3, 64.3,
61.4, 61.1, 58.8, 57.2, 56.8, 53.9, 53.7, 50.4, 41.7, 39.6, 35.5, 34.5,
33.2, 33.0, 31.2, 28.4, 28.3, 26.4, 25.6, 21.6, 21.3, 10.7. ESI-MS:
m/z 1461.7 [M+H]+, 1482.7 [M+Na]+.
4.15.2. Compound 18a
TFA (900 lL, 10 mmol) was added to a stirred solution of com-
pounds 16e (95 mg, 0.27 mmol) in CH2Cl2 (3 mL). The reaction
mixture was left stirring at room temperature and then concen-
trated under reduced pressure. The crude product was used in
the next reaction step without further purification.
HOBt (74.6 mg, 0.55 mmol), HBTU (208.6 mg, 0.55 mmol) and
sym-collidine (132.1 lL, 1 mmol) were sequentially added to a
stirred solution of the crude residue (theoretically 0.27 mmol)
and compound 4d (150 mg, 0.034 mmol) in dry DMF (3 mL) at
0 °C. The reaction mixture was left stirring and monitored by LC–
MS. After reaction completion, the mixture was diluted with EtOAc
(20 mL) and washed with 5% aqueous citric acid (20 mL), saturated
aqueous sodium bicarbonate (20 mL) and brine (2 ꢀ 20 mL). The
organic layer was dried over Na2SO4, and then the solvent was re-
moved under reduced pressure. The residue was purified by
Biotage™ flash chromatography, eluant conditions: from 1% of
MeOH and 99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield
82% (170 mg, MW 774.92, 0.220 mmol) of pure 18a. Analytical
4.15.5. Compound 10a
TFA (270 lL, 3.0 mmol) was added to a stirred solution of com-
pound 19a (54 mg, 0.030 mmol) in CH2Cl2 (5 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc

6706
L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
column, eluant conditions: from 40% of H2O (0.1% TFA) and 60% of
CH3CN (0.1% TFA) to 25% of H2O (0.1% TFA) and 75% of CH3CN (0.1%
TFA), flow rate 15 ml/min, 22 min runs. Yield 90% (41 mg, MW
1488.60, 0.027 mmol) of pure 10a. Analytical characterization: 1H
NMR (400 MHz, D2O): d: 8.77 (s, 1H), 7.39–7.29 (m, 15H), 6.04 (s,
1H), 5.33 (s, 1H), 4.67 (d, J = 10.0 Hz, 2H), 4.60–4.40 (m, 5H), 4.00
(m, 2H), 3.90 (m, 2H), 3.79 (m, 2H), 3.58 (d, J = 3.6 Hz, 2H), 3.55–
38 (m, 11H), 3.35–3.20 (m, 2H), 2.96 (m, 3H), 2.67 (s, 6H), 2.57
(t, J = 7.2 Hz, 2H), 2.23 (m, 2H), 2.12 (m, 2H), 2.07–1.85 (m, 8H),
1.85–1.62 (m, 7H), 1.55 (m, 1H), 1.50–1.30 (m, 2H), 0.95 (m, 6H);
13C NMR (100 MHz, D2O): d:174.8, 173.2, 172.7, 171.8, 171.1,
169.9, 168.4, 168.2, 146.0, 141.0, 140.8, 135.9, 129.2, 129.0,
128.8, 127.8, 127.3, 127.1, 123.9, 69.5, 69.4, 68.7, 68.6, 63.2, 62.7,
62.6, 61.7, 58.7, 58.3, 58.0, 57.6, 54.5, 54.4, 50.0, 40.7, 39.2, 38.9,
37.2, 35.1, 32.4, 31.5, 31.4, 30.1, 29.4, 27.9, 27.7, 23.4, 21.0, 8.2.
ESI-MS: m/z 631.3 [M+H]2+, 1260.8 [M+H]+, 1282.7 [M+Na]+.
4.16.3. Compound 19b
A 0.9 M aqueous solution of sodium ascorbate (60 lL) and a
0.3 M aqueous solution of Cu(OAc)2 (40 lL) were sequentially
added to a stirred solution of compound 19c (37 mg, 0.045 mmol)
and compound 4f (29 mg, 0.045 mmol) in a 1:1 mixture of
H2O/tBuOH (750
lL). The reaction mixture was stirred overnight
at room temperature and then the solvent was removed under re-
duced pressure. The residues were purified by Biotage™ flash chro-
matography, eluant conditions: from 1% of MeOH and 99% of
CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 80% (52 mg,
MW 1460.79, 0.036 mmol) of pure 19b. Analytical characteriza-
tion: 1H NMR (400 MHz, CDCl3): d: 7.89 (d, J = 6.8 Hz, 1H), 7.71
(d, J = 7.0 Hz, 1H), 7.47 (m, 1H), 7.35–7.12 (m, 15H), 7.08 (d,
J = 7.2 Hz, 1H), 6.85 (bs, 1H), 6.55 (bs, 1H), 6.10 (d, J = 8.8 Hz, 1H),
5.36 (d, J = 7.2 Hz, 1H), 4.56 (m, 3H), 4.38 (m, 4H), 4.10 (bs, 1H),
3.75 (m, 2H), 3.60–3.20 (m, 18H), , 2.97 (bs, 2H), 2.75 (s, 6H),
2.55 (m, 3H), 2.32 (m, 1H), 2.15 (m, 3H), 1.95–1.60 (m, 13H),
1.43 (s, 9H), 1.38 (s, 9H); 1.35–1.15 (m, 3H), 1.02 (m, 1H), 0.85 (t,
J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3): d: 171.9, 171.4, 170.7,
169.6, 141.8, 141.2, 138.4, 128.8, 128.7, 128.1, 127.6, 127.5,
127.3, 127.2, 127.1, 70.4, 70.1, 69.9, 69.8, 69.5, 64.3, 61.4, 61.3,
58.8, 57.2, 56.9, 53.9, 53.6, 41.6, 40.4, 39.6, 39.2, 34.4, 33.6, 33.3,
33.0, 31.1, 28.4, 28.3, 26.5, 25.9, 21.7, 21.2, 10.7. ESI-MS: m/z
681.2 [M+H]2+,1461.0 [M+H]+, 1483.0 [M+Na]+.
4.16. Head–tail dimer 10b—Schemes 12 and 14
4.16.1. Compound 18b
A 1.0 M aqueous citric acid solution (250 lL) was added to a
solution of 18a (170 mg, 0.220 mmol) in H2O/EtOH 1:9 (3 mL).
The solution was hydrogenated by flowing through a 10% Pd/C cat-
alyst cartridge (hydrogen pressure 10 bar, temperature 35 °C, flow
rate 0.5 mL/min) using the H-Cube™ system. After reaction com-
pletion, the solvent was removed under reduced pressure. The
crude product was used without any further purification, and only
an analytical sample was purified by semi-preparative HPLC re-
verse phase chromatography, eluant conditions: from 90% H2O
(1% CH3COOH) and 10% CH3CN (1% CH3COOH) to 100% CH3CN
(1% CH3COOH). Yield 98% (162 mg, MW 748.95, 0.219 mmol) of
pure 18b. Analytical characterization: 1H NMR (400 MHz, CD3OD):
4.16.4. Compound 10b
TFA (180 lL, 2.0 mmol) was added to a stirred solution of com-
pound 19b (30 mg, 0.022 mmol) in CH2Cl2 (4 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from 40% of H2O (0.1% TFA) and 60% of
CH3CN (0.1% TFA) to 25% of H2O (0.1% TFA) and 75% of CH3CN
(0.1% TFA), flow rate 15 ml/min, 22 min runs. Yield 86% (28 mg,
MW 1488.60, 0.019 mmol) of pure 10b. Analytical characteriza-
tion: 1H NMR (400 MHz, D2O): d: 7.78 (s, 1H), 7.37–7.28 (m,
15H), 6.04 (s, 1H), 5.34 (s, 1H), 4.78 (d, J = 10.4 Hz, 1H), 4.66 (m,
1H), 4.53 (m, 3H), 4.36 (m, 1H), 4.03 (m, 2H), 3.88 (t, J = 6.0 Hz,
2H), 3.60–3.25 (m, 16H), 3.28 (m, 3H), 2.94 (t, J = 14.4 Hz, 2H),
2.70 (s, 3H), 2.58 (s, 3H), 2.56 (t, J = 7.2 Hz, 2H), 2.35–2.05 (m,
5H), 2.05–1.88 (m, 9H), 1.85–1.65 (m, 6H), 1.62–1.46 (m, 3H),
0.95 (m, 6H); 13C NMR (100 MHz, D2O): d: 174.9, 173.2, 172.7,
171.1, 169.5, 168.7, 146.5, 141.0, 140.9, 136.0, 129.2, 129.0,
128.9, 127.8, 127.4, 127.3, 127.1, 69.6, 69.4, 68.8, 68.7, 63.2, 62.8,
62.6, 61.9, 61.8, 58.7, 58.2, 58.0, 57.6, 54.5, 54.4, 52.0, 39.3, 39.0,
38.1, 35.0, 32.4, 32.3, 31.8, 31.5, 27.9, 27.7, 23.5, 23.4, 20.9, 8.2,
8.1. ESI-MS: m/z 631.1 [M+H]2+, 1260.8 [M+H]+, 1282.7 [M+Na]+.
8.6 (bs, 1H), 7.44–7.33 (m, 5H), 5.40 (s, 1H), 4.67 (dd, J = 5.2,
d:
2.8 Hz, 1H), 4.53 (m, 2H), 4.01 (m, 1H), 3.67 (m, 5H), 3.65–3.47
(m, 8H), 3.39 (m, 2H), 3.09 (bs, 2H), 2.85 (s, 3H), 2.27 (m, 1H),
2.16 (m, 1H), 2.10- 1.88 (m, 4H), 1.82–1.65 (m, 4H), 1.55 (m, 1H),
1.50 (s, 9H), 1.42 (bs, 2H), 0.94 (t, J = 7.2 Hz, 3H); 13C NMR
(100 MHz, CD3OD): d: 172.3, 171.8, 171.0, 137.6, 128.4, 127.9,
127.2, 70.1, 69.8, 69.0, 67.4, 63.9, 61.4, 58.5, 57.3, 40.3, 39.6,
39.2, 33.2, 32.8, 30.7, 29.5, 27.3, 27.2, 26.9, 9.6. ESI-MS: m/z
749.6 [M+H]+, 771.7 [M+Na]+.
4.16.2. Compound 18c
DCC (24.8 mg, 0.12 mmol) and DMAP (3 mg, 0.025 mmol) were
sequentially added to a stirred solution of compound 18b (83 mg,
0.11 mmol) and 4-pentynoic acid (0.11 mmol) in dry CH2Cl2 (4 mL)
at 0 °C. The reaction mixture was warmed to room temperature
over a period of 1 h. After reaction completion, the reaction mix-
ture was filtered, and washed with diethyl ether. The combined
solvents were removed under reduced pressure, and the crude
product was purified by Biotage™ flash chromatography, eluant
conditions: : from 1% of MeOH and 99% of CH2Cl2 to 10% of MeOH
and 90% of CH2Cl2. Yield 74% (67 mg, MW 829.01, 0.081 mmol) of
pure 18c. Analytical characterization: 1H NMR (400 MHz, CDCl3):
d: 7.98 (d, J = 7.0 Hz, 1H), 7.51 (d, J = 6.8 Hz, 1H), 7.45–7.25 (m,
5H), 6.68 (bs, 1H), 5.43 (d, J = 7.2 Hz, 1H), 4.71 (d, J = 6.8 Hz, 1H);
4.48 (bs, 2H), 3.8 (m, 1H), 3.70- 3.30 (m, 18H), 2.87 (s. 3H), 2.48
(m, 2H), 2.40–2.15 (m, 4H), 2.15–1.65 (m, 9H), 1.52 (s, 9H), 1.40–
1.25 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d: 173.4, 173.2, 171.4, 171.3, 170.1, 169.5, 138.2, 128.8, 128.2,
127.2, 83.2, 70.6, 70.5, 70.4, 70.3, 70.1, 69.8, 69.5, 69.2, 64.3, 61.4,
58.8, 57.2, 53.9, 41.6, 39.7, 39.6, 39.1, 35.1, 34.5, 33.0, 31.2, 28.4,
26.6, 21.6, 20.6, 14.8, 10.6. ESI-MS: m/z 829.6 [M+H]+, 851.5
[M+Na]+.
4.17. Head–tail dimer 10c—Schemes 12 and 15
4.17.1. Compound 18d
Diglycolic anhydride (25.5 mg, 0.22 mmol) and pyridine
(36.4 lL, 0.45 mmol) were sequentially added to a stirred solution
of compound 18b (83 mg, 0.11 mmol) in DMF (3 mL). The reaction
mixture was stirred at room temperature for 4 h. After reaction
completion, water (20 mL) was added to the reaction mixture.
The mixture was diluted with EtOAc (20 mL) and washed with
5% aqueous citric acid (20 mL), saturated aqueous sodium bicar-
bonate (20 mL) and brine (2 ꢀ 20 mL). The combined organic layer
was dried over Na2SO4, and then the solvent was removed under
reduced pressure. Finally, the crude product was purified by flash
chromatography on a Biotage™ C18 reverse phase column, eluant
conditions: from 90% H2O (1% CH3COOH) and 10% CH3CN (1%
CH3COOH) to 100% CH3CN (1% CH3COOH). Yield 58% (55 mg, MW
865.00, 0.064 mmol) of pure 18d. Analytical characterization: 1H

L. Manzoni et al. / Bioorg. Med. Chem. 20 (2012) 6687–6708
6707
NMR (400 MHz, CDCl3): d: 8.01 (d, J = 7.0 Hz, 1H), 7.61 (bs, 1H),
7.53 (d, J = 6.8 Hz, 1H), 7.40–7.28 (m, 5H), 6.98 (bs, 1H), 4.52 (d,
J = 7.2 Hz, 1H), 4.71 (d, J = 6.8 Hz, 1H), 4.5 (m, 2H), 4.10 (m, 3H),
3.82 (m, 1H); 3.70–3.30 (m, 18H), 2.88 (s, 3H), 2.25 (m, 2H), 2.08
(s, 2H), 2.05–1.70 (m, 8H), 1.51 (s, 9H), 1.39 (m, 2H), 0.93 (t,
J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d: 175.2, 173.0, 171.9,
171.4, 169.8, 169.8, 137.9, 128.8, 128.3, 127.2, 77.3, 77.0, 76.8,
71.4, 70.4, 70.0, 69.7, 69.5, 69.2, 64.3, 61.6, 58.9, 57.2, 54.0, 41.5,
39.7, 38.8, 34.3, 33.0, 31.2, 28.4, 26.7, 21.6, 20.6, 10.6. ESI-MS: m/
z 865.4 [M+H]+, 887.4 [M+Na]+.
added to A stirred solution of compound 4e (61.8 mg, 0.10 mmol)
and of the alkyne 20a55 (70.8 mg, 0.10 mmol) in a 1:1 mixture of
H2O/tBuOH (300
lL). The reaction mixture was stirred overnight
at room temperature, and then the solvent was removed under
reduced pressure. Finally, the residue was purified by flash chro-
matography, eluant conditions: isocratic, CH2Cl2/MeOH 95/5. Yield
57% (76 mg, MW 1324.62, 0.057 mmol) of pure 19d. Analytical
characterization: ½a 2D0
ꢁ
ꢂ55.6 (c 0.54, MeOH); 1H NMR (400 MHz,
CDCl3): d: 7.83 (d, J = 7.2 Hz, 1H), 7.76 (d, J = 7.2 Hz, 2H), 7.57 (m,
5H), 7.49 (bd, 1H), 7.45–7.13 (m, 18H), 6.17 (d, J = 8.2 Hz, 1H),
5.23 (bd, J = 7.2 Hz, 1H), 4.80–4.30 (m, 9H), 4.30–3.90 (m, 6H),
3.90–3.50 (m, 4H), 2.87 (s, 3H), 2.38 (m, 1H), 2.21 (m, 1H), 2.02
(m, 1H), 1.94–1.45 (m, 10H), 1.43–1.32 (m, 11H), 1.30–1.05 (m,
6H), 0.93 (t, 3H); 13C NMR (100 MHz, CDCl3): d: 173.0, 171.2,
170.7, 169.4, 169.3, 155.7, 153.3, 143.9, 141.8, 141.3, 133.0,
129.8, 128.8, 127.7, 127.4, 127.3, 127.2, 127.1, 125.0, 120.0, 80.1,
74.6, 68.0, 62.6, 61.2, 58.7, 56.8, 56.3, 54.9, 54.6, 53.7, 51.8, 48.4,
47.7, 47.2, 40.4, 33.5, 33.2, 31.1, 28.5, 27.2, 26.0, 25.6, 24.0, 16.3,
10.2. ESI-MS: m/z 1325.4 [M+H]+, 1347.4 [M+Na]+.
4.17.2. Compound 19c
HOBt (9.5 mg, 0.078 mmol), HBTU (29.6 mg, 0.078 mmol) and
sym-collidine (33 lL, 0.25 mmol) were sequentially added to a
stirred solution of compound 18d (54 mg, 0.063 mmol) and com-
pound 4h (52 mg, 0.08 mmol) in dry DMF (1 mL) at 0 °C. The reac-
tion mixture was left stirring and monitored by LC–MS. After
reaction completion, the mixture was diluted with EtOAc (20 mL)
and washed with 5% aqueous citric acid (20 mL), saturated aque-
ous sodium bicarbonate (20 mL) and brine (2 ꢀ 20 mL). The organ-
ic layer was dried over Na2SO4, and then the solvent was removed
under reduced pressure. The residue was purified by Biotage™
flash chromatography, eluant conditions: from 1% of MeOH and
99% of CH2Cl2 to 10% of MeOH and 90% of CH2Cl2. Yield 67%
(61 mg, MW 1457.77, 0.042 mmol) of pure 19c. Analytical charac-
terization: 1H NMR (400 MHz, CDCl3): d: 7.87 (d, J = 7.2 Hz, 1H),
7.76 (d, J = 6.8 Hz, 1H), 7.63 (d, J = 6.0 Hz, 1H), 7.46 (m, 2H),
7.35–7.10 (m, 15H), 6.72 (bs, 1H), 6.15 (d, J = 8.4 Hz, 1H), 5.35 (d,
J = 7.2 Hz, 1H), 4.64 (dd, J = 19.2, 7.2 Hz, 2H), 4.40 (m, 4H), 3.95
(m, 4H), 3.72 (m, 3H), 3.6–3.20 (m, 18H), 2.79 (s, 3H), 2.75 (s,
3H), 2.52 (m, 2H), 2.35 (m, 1H), 2.18 (m, 2H), 1.90–1.55 (m,
14H), 1.43 (s, 9H), 1.41 (s, 9H), 1.30–1.10 (m, 4H), 0.85 (m, 6H);
13C NMR (100 MHz, CDCl3): d: 172.7, 171.3, 169.6, 168.9, 168.9,
142.2, 138.4, 128.8, 128.6, 128.5, 128.1, 127.4, 127.3, 127.2, 70.7,
70.5, 70.4, 70.3, 70.1, 69.6, 69.5, 64.3, 61.7, 61.4, 61.3, 60.5, 58.9,
58.8, 57.2, 56.8, 53.9, 53.5, 41.6, 40.0, 39.7, 39.6, 38.7, 34.4, 34.3,
33.1, 33.0, 31.1, 28.4, 28.3, 26.4, 25.7, 21.7, 21.3, 10.7, 10.6. ESI-
MS: m/z 1452.9 [M+H]+, 1475.0 [M+Na]+.
4.18.2. Compound 10d
Compound 19d (59.7 mg, 0.045 mmol) was dissolved into a 20%
solution of piperidine in CH2Cl2 (2 mL). The reaction mixture was
stirred for 30 min at room temperature. and then concentrated in
vacuo. The residue was used as such in the next reaction step.
A 3 N solution of HCl in MeOH (0.5 mL) was added to a stirred
solution of crude (theoretically 0.045 mmol) in MeOH (2 mL). The
reaction mixture was left stirring at room temperature overnight
and then concentrated under reduced pressure. The residue was
purified by chromatography on a C18 reverse phase semi-prepara-
tive hplc column, eluant conditions: from 80% of H2O (0.1% TFA)
and 20% of CH3CN (0.1% TFA) to 50% of H2O (0.1% TFA) and 50%
of CH3CN, flow rate 20 ml/min, 10 min runs. Yield 90% (45 mg,
MW 1231.29, 0.040 mmol) of pure 10d. Analytical characteriza-
tion: ½a D20
ꢁ
ꢂ29.0 (c 0.88, H2O); 1H NMR (400 MHz, D2O): d: 7.86
(s, 1H); 7.43–7.05 (m, 15H), 5.91 (s, 1H), 4.63–4.10 (m, 10H),
3.95–3.80 (m, 4H), 3.48 (m, 2H), 2.59 (s, 3H), 2.25–1.38 (m, 16H),
1.23 (m, 2H), 1.14 (m, 3H), 0.96 (m, 3H); 13C NMR (100 MHz,
D2O): d: 172.0, 169.7, 169.5, 154.7, 144.5, 141.2, 141.0, 134.0,
133.3, 130.2, 130.0, 128.7, 127.6, 127.3, 127.0, 126.6, 125.2, 74.0,
61.5, 58.1, 57.4, 57.0, 56.5, 56.0, 54.3, 54.1, 51.8, 48.9, 47.6, 38.6,
32.3, 31.0, 29.6, 28.6, 27.7, 26.9, 24.4, 23.5, 16.0, 15.3, 8.4. ESI-
4.17.3. Compound 10c
TFA (360 lL, 4.0 mmol) was added to a stirred solution of com-
pound 19c (58 mg, 0.040 mmol) in CH2Cl2 (5 mL). The reaction
mixture was left stirring at room temperature overnight and then
concentrated under reduced pressure. The residue was purified by
chromatography on a C18 reverse phase semi-preparative hplc col-
umn, eluant conditions: from 40% of H2O (0.1% TFA) and 60% of
CH3CN (0.1% TFA) to 25% of H2O (0.1% TFA) and 75% of CH3CN
(0.1% TFA), flow rate 15 ml/min, 22 min runs. Yield 88% (55 mg,
MW 1480.57, 0.037 mmol) of pure 10c. Analytical characteriza-
tion: 1H NMR (400 MHz, D2O): d: 7.41–7.30 (m, 15H), 6.06 (s,
1H), 5.36 (s, 1H), 4.67 (d, J = 8.4 Hz, 2H), 4.55 (m, 2H), 4.10 (d,
J = 5.2 hz, 4H), 4.05 (m, 2H), 3.93 (t, J = 6.4 Hz, 1H), 3.89 (t,
J = 5.6 Hz, 1H), 2.70 (s, 3H), 2.69 (s, 3H), 2.25 (m, 2H), 2.15 (m,
2H), 2.08–1-68 (m, 17H), 1.65–1.50 (m, 3H), 0.97 (m, 6H) ; 13C
NMR (100 MHz, D2O): d: 172.8, 171.8, 171.7, 171.1, 170.1, 168.5,
168.3, 141.0, 140.9, 129.2, 129.0, 128.9, 127.8, 127.4, 127.3,
127.2, 70.0, 69.9, 69.6, 69.4, 68.8, 68.7, 63.2, 62.8, 62.7, 61.9,
61.8, 58.8, 58.5, 58.0, 57.7, 54.6, 54.5, 40.7, 39.3, 39.0, 38.6, 37.4,
32.4, 31.6, 31.5, 30.5, 29.5, 29.0, 27.9, 27.7, 23.4, 8.2, 8.1. ESI-MS:
m/z 627.1 [M+H]2+, 1252.8 [M+H]+, 1274.8 [M+Na]+.
MS: m/z 1025.7 [M+Na]+, 502.4 [M+2H]2+
.
Acknowledgment
The authors are thankful to ‘FONDAZIONE CARIPLO’ for financ-
ing through Project 2009-2534, titled ‘Inhibitors of Apoptosis Pro-
teins (IAPs) as anticancer therapeutics’.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.09.020.
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