Design and synthesis of all diastereomers of cyclic pseudo-dipeptides as mimics of cyclic CXCR4 pentapeptide antagonists

Article abstract of DOI:10.1039/b702649h

The four diastereomers of 2,5-bis[(3-guanidino)propyl]-1-[3-(4- hydroxyphenyl)propionyl]-7-(2-naphthylacetyl)-1,4,7-triazacycloundec-9-en-3-one (54-57) and of 2,5-bis[(3-guanidino)propyl]-1-(4-hydroxyphenylacetyl)-7-(2- naphthylacetyl)-1,4,7-triazacycloundec-9-en-3-one (58-61) were synthesized by a divergent methodology from l- and d-glutamic acids. The 11-membered ring core was made by ring closing metathesis of linear bis(allylamines), and the guanidyl functions were introduced by a simultaneous double Mitsunobu reaction using bis(Boc)guanidine. These compounds were designed to mimic cyclic pentapeptide FC131 (c[Gly-d-Tyr-Arg-Arg-Nal]). The Royal Society of Chemistry.

Full text of DOI:10.1039/b702649h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Design and synthesis of all diastereomers of cyclic pseudo-dipeptides as
mimics of cyclic CXCR4 pentapeptide antagonists†
Je´roˆme Cluzeau,a Shinya Oishi,a Hiroaki Ohno,a Zixuan Wang,b Barry Evans,b Stephen C. Peiperb and
Nobutaka Fujii*a
Received 23rd February 2007, Accepted 24th April 2007
First published as an Advance Article on the web 14th May 2007
DOI: 10.1039/b702649h
The four diastereomers of 2,5-bis[(3-guanidino)propyl]-1-[3-(4-hydroxyphenyl)propionyl]-7-(2-
naphthylacetyl)-1,4,7-triazacycloundec-9-en-3-one (54–57) and of 2,5-bis[(3-guanidino)propyl]-
1-(4-hydroxyphenylacetyl)-7-(2-naphthylacetyl)-1,4,7-triazacycloundec-9-en-3-one (58–61) were
synthesized by a divergent methodology from L- and D-glutamic acids. The 11-membered ring core was
made by ring closing metathesis of linear bis(allylamines), and the guanidyl functions were introduced
by a simultaneous double Mitsunobu reaction using bis(Boc)guanidine. These compounds were
designed to mimic cyclic pentapeptide FC131 (c[Gly-D-Tyr-Arg-Arg-Nal]).
Introduction
CXCR4 chemokine receptor is involved in HIV-1 infection of T
cells. Attachment of virus envelope glycoprotein gp120 onto cell
surface proteins CD4 and CXCR4 leads to membrane fusion and
subsequent virus entry into the cell.1,2 Thus, CXCR4 is considered
an important therapeutic target. Several potent CXCR4 antago-
nists have been developed so far. Among these, we have previously
identified a b-sheet-like 14-residue peptide3 T140, and its down-
sized analogues, cyclic pentapeptide FC1314 (c[Gly-D-Tyr-Arg-
Arg-Nal]) as potent and specific CXCR4 antagonists. These were
characterized as HIV-1 entry inhibitors (Fig. 1).5 Several other
non-peptidic, low-molecular-weight CXCR4 antagonists have also
been reported to inhibit HIV-1 infection through CXCR4, such
as KRH27316 and AMD3100.7 AMD3100 was abandoned as an
anti-HIV drug8 because of a lack of in vivo efficacy and undesirable
side effects; nevertheless, AMD070, an orally-available derivative
of AMD3100 has recently been described to be as potent as
AMD3100, and it will be further investigated as an HIV drug
in clinical trials.9,10
The use of constrained peptides or peptide mimics has become
popular for increasing receptor affinity, for the development of new
drugs, to investigate bioactive conformations, and to increase du-
ration of action. On native peptides, constraints can be introduced
by linkage between two points of the peptide (NH to NH,11 NH
to CO2H12 or disulfide bridge13) to fix a large secondary structure,
generally a helix or sheets. The small secondary structures (turn,
small helix, hairpin and E- or Z-amide bonds) can also be induced,
stabilized or fixed by introduction in the sequence of one or several
small constrained (Freidinger lactams14 or azabicycloalkanes15,16),
bulky (alkylprolines17) or rigid (dipeptide isosteres18,19) unnatural
aGraduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-
Fig. 1 Down-sizing CXCR4 antagonists.
ku, Kyoto, 606-8501, Japan. E-mail: nfujii@pharm.kyoto-u.ac.jp; Fax: +81
(0)75 753 4570; Tel: +81 (0)75 753 4551
amino acids. Finally, small peptides or parts of peptides can
be converted to small semi-peptidic or peptide-like structures,
synthesized to mimic peptide backbones, and possessing all the
necessary functional groups for activity.20
bDepartment of Pathology and Immunotherapy Center, Medical College of
Georgia, Augusta, Georgia, 30912, USA
† Electronic supplementary information (ESI) available: Experimental and
analytical data. See DOI: 10.1039/b702649h
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In our research on the development of new CXCR4 receptor
antagonists, we are looking for compounds with a lower molecular
weight than our current active peptide FC131, and with a more
stable and active structure. Our previous studies on FC131 led us
to modify its sequence and to use a linkage between two points
of the peptide by replacing Gly1 by an alkyl chain or disulfide
bridge.13a Unfortunately these modifications resulted in significant
loss of activity. Unnatural amino acids were also introduced to
the peptide sequence to perform structure–activity relationship
studies on the peptide backbone. For example, the replacement
of Arg4 by constrained amino acids cis- or trans-4-guanidino-Pro
furnished peptides with similar activities to that of the parent pep-
tide, showing the importance of the constrained structure of the
peptide backbone.13a On the other hand, introduction of E-alkene
dipeptide isosteres in positions 4–5 (Arg–Nal) and 5–1 (Nal–
Gly) led to peptides with similar pharmacophore orientations and
distances, and revealed the importance of amide bonds on the
backbone of the peptide.19 We are also interested in the synthesis
of small semi-peptidic compounds containing pharmacophores of
FC131. We have recently reported that a 3,6-dihydropyridin-2-one
analog containing two guanidine residues and a naphthyl group
retains moderate activity as a CXCR4 antagonist.21 We are now
interested in a less-rigid backbone structure, on which it would be
easy to vary side-chain length and chirality. Design of compounds
of type A was performed by removing Gly from the parent peptide
and making a linkage between the nitrogens (bold) of Arg and Nal
(Fig. 1). Such a structure could possess a propionyl or acetyl (n,
m = 0 or 1) side chain to replace tyrosine and naphthylalanine, and
propenyl linkage between nitrogens 1 and 7 to constrain dipeptides
and form 11-membered ring compounds.
Scheme 1 Retrosynthesis of compounds A.
Retrosynthetically, the ring can be closed by ring closing
metathesis (RCM) between two allylamine residues. RCM has
been shown to be a useful method for synthesizing constrained
peptides and forming 8–10-membered rings20a,22 and 13–20-
membered ring cycles.11,12,20b,c,d Nevertheless, the only reported
example of RCM on an 11-membered, ring-constrained peptide
failed,11 but we expected that cyclization would be possible with
a new generation of catalysts. Cyclization of these peptides was
reported to be possible without protection of the amide bond to
increase cis conformation for cycles of up to 10-membered rings.
To simplify the synthesis, the two nucleophilic guanidyl groups
should be introduced in the last steps by the Mitsunobu reaction
with corresponding alcohols (Scheme 1). Our two fragments
were N-substituted 5-hydroxynorvaline acids 3 and diamines 4.
Both of these could be synthesized from the same precursor,
L- or D-glutamic acid, to give easy access to the four possible
diastereomers.
Scheme 2 Synthesis of protected L- and D-5-hydroxynorvaline.
the fully protected L-5-hydroxynorvaline L-6 at a yield of 46% (82%
brsm), and the D-5-hydroxynorvaline D-6 in a similar manner and
yield. Both amino acids 6 were obtained as inseparable mixtures
with TIPSOH, but this alcohol did not interfere with the next steps,
and yields were estimated by comparing 1H-NMR integration of
TIPS and other signals.
For the synthesis of diamines L- and D-4, esters 6 were
reduced to alcohols 7 using DIBAl-H in THF (Scheme 3). Pro-
tected amines 8 were obtained by the Mitsunobu reaction be-
tween alcohols 7 and ortho-nitrobenzenesulfonyl allylamine (Ns-
allylamine) 9, with moderate yields. Boc removal to form both
amines L- and D-4 in TFA–DCM (2 : 3) were carried out just
before coupling with acids 3.
For the synthesis of acids 3, conversion of Boc carbamate to allyl
amine was carried out on a small scale with a two-step procedure.
Boc removal with TFA and selective mono-allylation of the free
amine using the Kim protocol gave allyl amine 10.26 Unfortunately,
we were not able to obtain a good yield of monoallylamine on the
Results and discussion
Synthesis of 5-hydroxynorvaline, also called pentahomoserine, has
been reported many times in protected and unprotected forms.23
We chose to use the procedure described by Kokotos24 from
glutamic acid (Scheme 2). L- and D-Glu were efficiently mono-
benzylated using benzyl alcohol, H2SO4 and pyridine;25 the amine
was protected with Boc; the a-acid was converted to a methyl ester,
and the benzyl ester was removed by hydrogenation with Pd/C to
give the free acids 5, on a large scale. Reduction of acids to alcohols
using the mixed anhydrides and protection with TIPSCl provided
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Table 1 Amino acid coupling of compounds 3 and 4
Acid
Amine
R
Products (yield)
1
2
3
L-3a
L-3a
L-3b
L-4
D-4
L-4
BnOPh(CH2)2
BnOPh(CH2)2
BnOPhCH2
14/15 (88%)
16/17 (91%)
18 (43%)
19 (48%)
4
L-3b
D-4
BnOPhCH2
20 (37%)
21 (53%)
Scheme 3 Synthesis of amines 4.
5 g scale (35%) by this method, and diallylamine became the major
product. We decided to use a longer but more reliable method using
an ortho-nitrobenzenesulfonyl (Ns) protecting group (Scheme 4).
Boc carbamate was removed with TFA and replaced by a nosyl
group by reaction with NsCl and Et3N in DMF. Nosyl amine
was easily allylated using allyl bromide and potassium carbonate
in DMF, and the nosyl group was removed using mercaptoacetic
acid under basic conditions to give 10, with a 63% yield, from 6 on
a 5 g scale. Allylamine 10 was then coupled with acids 11 and
12 using O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU)27 in DMF to give allylamides 13a
and 13b, with 95 and 86% yields, respectively. Saponification of
the methyl esters with LiOH furnished the desired acids L-3a and
L-3b.28
Scheme 5 Synthesis of dienes 14 to 21.
with amine D-4 using the same conditions and gave a mixture
of 16 and 17. Acid L-3b was coupled with amines L-4 and D-4 to
give separable mixtures of diastereomers 18/19 and 20/21 in good
yields.
RCM of allylamines has been reported to react differently
depending on the temperature and the catalyst used. Grubb’s I and
Grubb’s II catalysts have been reported to lead to partial or total
isomerization of the olefin, depending on reaction temperature
and substrates, to give cyclic or acyclic enamides.22e,29 This
isomerization process does not seem to be general, indeed normal
reactivity has also been reported on similar substrates with both
catalysts.30 We first attempted RCM on the diene mixture 14/15
using 0.2 eq. of Grubb’s catalysts of first and second generation, in
CH2Cl2 at refluxandhigh dilution(6to3 mM, Scheme 6). Reaction
with Grubb’s I catalyst proceeded slowly and was not completed
within 24 h. A low yield of the desired separable 11-membered
ring diastereomers 22 and 23 was nevertheless obtained (Table 2,
entry 1). Surprisingly, in contrast to the reported observation
of RCM with allylamines, reaction with Grubb’s II catalyst was
completed after 12 h and gave a good yield of diastereomers 22 and
23. (Table 2, entry 2). The structures of 22 and 23 were confirmed
using COSY NMR spectra of 22 and 39 (derived from 23). Both
Table 2 RCM of linear dienes
Scheme 4 Synthesis of acids 3a and 3b.
Diene
n
Grubb’s catalyst
Product (yield)
The amino acid coupling of acid L-3a and amine L-4 proceeded
completely using HATU, 1-hydroxy-7-azabenzotriazole (HOAt)
and DIEA in DMF, but gave a 1 : 1 mixture of two inseparable
diastereomers, 14 and 15 (Scheme 5, Table 1, entry 1). We were
not able to suppress epimerization of the acid during the coupling
step. We decided to use this epimerization to our advantage for the
synthesis of the different diastereomers. Acid L-3a was coupled
1
2
3
4
5
6
7
14/15
14/15
16/17
18
1
1
1
0
0
0
0
I
22 (13%) + 23 (25%)
22 (43%) + 23 (42%)
24 (38%) + 25 (35%)
26 (69%)
II
II
II
II
II
II
19
27 (65%)
20
21
28 (71%)
29 (67%)
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Scheme 6 RCM of diallylamines 14–21 to 11-membered rings 22–29.
compounds had the desired but-2-ene-1,4-diamine structure, but
enamide structures coming from olefin isomerization anticipated
for the reaction with Grubb’s II catalyst at high temperature were
not observed.31
The diastereomeric mixture 16/17 and the single diastereomers
18–21 were then cyclized using Grubb’s II catalyst in DCM at
40 ◦C, to give products 24–29 in good yields. All the compounds
appeared to have E-olefins; Z-olefins were not observed.
Scheme 7 Introduction of naphthylacetyl side chain and removal of
TIPS protecting groups. Expected structure of compound 39 using NOE
correlation.
Introduction of naphthylacetyl side chains was easily achieved
by removal of nosyl groups using mercaptoacetic acid under
basic conditions on cycles 22–29 to form the free amines, which
were directly acylated with naphthylacetic acid using HATU and
triethylamine, to give compounds 30–37 (Scheme 7, Table 3). These
compounds were then submitted to tetrabutylammonium fluoride
(TBAF) solution at −20 ◦C to remove both TIPS groups. The
first TIPS group was completely removed in less than 24 h (by
TLC), but removal of the second TIPS appeared to be very slow
and needed a large excess of TBAF. Increasing the temperature
up to −20 ◦C accelerated the rate of deprotection but led to
formation of unidentified side produc◦ts on TLC and coloration
of the solution. After 72–96 h at −20 C with 6 eq. TBAF, diols
38–45 were isolated in moderate to good yields. The structures
and relative stereochemistry of diastereomers were established
based on the known stereochemistry at carbon 5 and one- and
two-dimensional NMR experiments. The majority of the NMR
signals were well resolved at distinct chemical shifts for product
39. All protons were assigned using the COSY experiment, which
established their linear sequence around the macrocycle. A large
coupling constant for NH proton (JNH 9.5 Hz),32 indicating a
dihedral angle of −130◦ or −110◦, and observation in the NOESY
spectra of a correlation between C2 proton and NH allowed us
to attribute the (2R,5S) stereochemistry to compound 39, and
therefore to other diastereomers. Unfortunately, we were not able
to get clearer proof of the stereochemistry at C2.
Introduction of a guanidyl group via the Mitsunobu reac-
tion can be achieved using bis- or tris-Boc- or Cbz-guanidine
(Scheme 8).23c,33 We decided to use the bis-Boc-guanidine for the
acidic cleavage of the Boc group and because bis-Boc has only one
reactive site while tris-Boc has the possibility to react twice. The
reaction worked well with simple alcohols, with yields up to 90%,33c
but gave average results on amino acids, with yields of 40–63%. In
Table 3 Introduction of naphthyl side chain and TIPS deprotection
Ns amine
R
Acyl product (yield, 2 steps)
Diol (yield)
1
2
3
4
5
6
7
8
(2S,5S)-22
(2R,5S)-23
(2R,5R)-24
(2S,5R)-25
(2S,5S)-26
(2R,5S)-27
(2R,5R)-28
(2S,5R)-29
BnOPh(CH2)2
BnOPh(CH2)2
BnOPh(CH2)2
BnOPh(CH2)2
BnOPhCH2
BnOPhCH2
BnOPhCH2
30 (63%)
31 (80%)
32 (44%)
33 (65%)
34 (63%)
35 (66%)
36 (58%)
37 (68%)
38 (78%)
39 (74%)
40 (72%)
41 (72%)
42 (59%)
43 (98%)
44 (89%)
45 (78%)
BnOPhCH2
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taining a D configuration for the second Arg, showed no activity.
This confirms the importance of this chiral center for antagonist
activity. The four compounds containing the less flexible phenol
side chain, 58–61, lost all antagonist activity.
Conclusion
We report here the divergent synthesis of tetra-substituted 11-
membered ring compounds 54–61 from L- and D-glutamic acids.
Two of these compounds, 54 and 55, showed antagonist activity
against CXCR4 receptors, and could be potential scaffolds for the
development of novel low-molecular-weight CXCR4 antagonists.
Experimental
Methyl (S)-2-(N-Boc-amino)-5-(triisopropylsilyloxy)pentanoate
(L-6) [Boc-Hnv(TIPS)-OMe]
Methyl (S)-2-(N-Boc-amino)-5-hydroxypentanoate (18.4 g,
74.5 mmol) was dissolved in DMF (100 ml) and was treated with
triethylamine (20.8 ml, 2 eq.) and TIPSCl (19.1 ml, 1.2 eq.) and
stirred for 5 h at rt. The reaction was partitioned between ether
and HCl 0.5 M. The organic layer was washed twice with HCl
(0.5 M). The combined aqueous layers were extracted with ether.
The combined ether fractions were washed with water and brine,
dried over MgSO4 and concentrated. The oil was purified by flash
chromatography using 90 : 10 hexane–ethyl acetate to afford an
inseparable mixture (3 : 1) of L-6 and TIPSOH (29.9 g, 87%). 1H
NMR (CDCl3) d 5.20 (d, 1H, J = 7.8 Hz), 4.30 (q, 1H, J = 7.5 Hz),
3.73 (s, 3H), 3.70 (t, 2H, J = 6.3 Hz), 1.90 (m, 1H), 1.75 (m, 1H),
Scheme 8 Synthesis of final compounds.
our case, the double reaction occurred, but products 46–53 were
isolated as inseparable mixtures with triphenylphosphine oxide.
The reaction was not completed in the case of products 38 and
41 (Table 4, entries 1 and 4). Starting materials were recovered
and resubmitted to Mitsunobu conditions. Enantiomers 42 and
44 were not soluble in THF, so the reaction was carried out in
THF–toluene–DMF (6 : 1 : 1) solutions (Table 4, entries 5 and 7).
The mixtures of products and Ph3PO were directly submitted to
acidic cleavage of the protecting groups. All protecting groups,
including benzyl ether, could be cleaved in acidic conditions
using TFA–CH2Cl2 (3: 1), with a large excess of anisole and
pentamethylbenzene as scavengers of benzyl cations.34 Products
54–61 were purified by semi-preparative HPLC and were isolated
in 9–40% yields from diols 38–45.
These compounds were tested as CXCR4 receptor antagonists
using inhibition of [125I]-SDF-1 binding to CXCR4 transfectants.
Compounds 54 and 55 retained activity against CXCR4 receptor
(IC50 3.4 and 3.2 lM), despite significant loss of activity when
compared to the native peptide (FC131, IC50 0.004 lM).44 As
observed in cyclic pentapeptides, diastereomers 56 and 57, con-
1.59 (m, 2H), 1.44 (s, 9H), 1.06 (m, 27 H (21H + TIPSOH)). 13
C
(CDCl3) d 174.3, 156.2, 80.1, 62.8, 53.6, 52.4, 29.2, 28.8, 28.4, 18.0,
17.8, 12.3, 12.0. FTIR (cm−1) 2941, 2865, 1717, 1503, 1462, 1365,
1166, 1104, 1012, 881. MS (FAB+) : m/z = 404 (M + H+).
(S)-2-(N-Boc-amino)-5-(triisopropylsilyloxy)pentan-1-ol (L-7).
The 3 : 1 mixture of L-6 and TIPSOH (10 g, 24.8 mmol of 6) was
dissolved in THF (250 ml) and cooled down to −5 ◦C. DIBAl-H
1M in toluene (87 ml, 4 eq.) was added dropwise over 20 min. The
reaction was stirred for an hour at 0 ◦C. The reaction was quenched
by AcOH (5 ml), diluted with HCl 0.5 M (200 ml) and extracted
3 times with EtOAc (100 ml). The combined organic layers were
washed with brine, dried over MgSO4 and concentrated. The oil
was purified by flash chromatography using hexane–EtOAc (80 :
20 to 50 : 50) to furnish L-7 (6.46 g, 79%). 1H NMR (CDCl3) d 4.89
Table 4 Introduction of guanidyl groups and final deprotection
Diol
n
Final product
Yield (2 steps)
m/z (calcd), (MH+)
m/z (MH+)
1
2
3
(2S,5S)-38
(2R,5S)-39
(2R,5R)-40
(2S,5R)-41
(2S,5S)-42
(2R,5S)-43
(2R,5R)-44
(2S,5R)-45
1
1
1
1
0
0
0
0
54
55
56
57
58
59
60
61
9%
684.3986
”
”
”
684.3984
684.3992
684.3993
684.3973
670.3822
670.3821
670.3820
670.3835
19%
26%
10%
40%
19%
27%
15%
4
5a
6
670.3829
”
”
”
7a
8
a Mitsunobu reaction was done in THF–toluene–DMF (6 : 1 : 1).
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(br s, 1H), 3.70–3.56 (m, 5H), 2.71 (br s, 1H), 1.62 (m, 3H), 1.44
(m, 10H), 1.06 (m, 21H). 13C (CDCl3) d 157.6, 80.0, 66.7, 63.2,
53.1, 29.3, 28.5, 27.8, 18.1, 12.0. FTIR (cm−1) 3339, 2942, 2866,
1687, 1506, 1461, 1390, 1365, 1246, 1170, 1102, 881. [a]2D4 −7.9
(c = 5.0, CHCl3), MS (FAB+) m/z 376 (M + H+); HRMS calcd
for C19H42NO4Si+ (MH+) 376.2883, found 376.2887.
toluene (60 ml) and DCM was removed under reduced pressure.
To the solution was carefully added saturated NaHCO3 solution.
The aqueous layer was extracted twice with ethyl acetate. The com-
bined organic fractions were washed with brine, dried over MgSO4
and concentrated. The oil was used without further purification.
The free amine was dissolved in DCM (20 ml) and was treated with
Et3N (1 ml, 1.2 eq.) followed by nosyl chloride (2.92 g, 1.1 eq.)
in solution in DCM (20 ml). The reaction was stirred overnight
and was then washed twice with 10% citric acid solution, with
brine, dried over MgSO4 and concentrated. The oil was purified
by flash chromatography using hexane–EtOAc (90 : 10 to 60 :
40) to furnish methyl (S)-2-[N-(o-nitrobenzenesulfonyl)amino)-
5-(triisopropylsilyloxy]pentanoate [Ns-Hnv(TIPS)-OMe] (4.95 g,
85%). 1H NMR (CDCl3) d 8.07 (m, 1H), 7.92 (m, 1H), 7.73
(m, 2H), 6.10 (d, 1H, J = 9.0 Hz), 4.22 (ddd, 1H, J = 5.4 Hz,
J = 8.0 Hz, J = 13.2 Hz), 3.70 (t, 2H, J = 6.1 Hz), 3.47 (s,
3H), 1.99 (m, 1H), 1.82 (m, 1H), 1.63 (m, 2H), 1.05 (m, 21H).
13C (CDCl3) d 171.6, 136.5, 133.6, 132.9, 130.5, 125.6, 125.3,
62.1, 56.6, 52.3, 29.9, 28.4, 18.0, 12.0. FTIR (cm−1) 2943, 2866,
1742, 1542, 1441, 1358, 1170, 1104.MS (FAB+) m/z 489 (M +
H+); HRMS calcd for C21H37N2O7SSi+ (MH+) 489.2085, found
489.2096. Nosyl amide (4.90 g, 10 mmol) was dissolved in DMF
(40 ml). Allyl bromide (1.3 ml, 1.5 eq.) and K2CO3 (1.80 g,
1.3 eq.) were successively added and the reaction was stirred
for 3 h at rt. The reaction was partitioned between ether and
water. The aqueous phase was extracted twice with ether. The
combined organic phases were successively washed with water
and brine, dried over MgSO4 and concentrated. The oil was
purified by flash chromatography using hexane–EtOAc (90 : 10 to
80 : 20) to furnish methyl (2S)-2-[N-(o-nitrobenzenesulfonyl)-N-
N-Allyl(o-nitrobenzene)sulfonamide. Nosyl chloride (5.54 g,
25 mmol) was dissolved in DCM (25 ml) and was cooled down
to 0 ◦C. Allylamine (4.7 ml, 62.5 mmol) was added dropwise.
The reaction was stirred at rt for 2 h. The solution was washed
twice with 10% citric acid solution, with brine, dried over MgSO4
and concentrated to give pure N-allyl(o-nitrobenzene)sulfonamide
(4.42 g, 73%). 1H NMR (CDCl3) d 8.13 (m, 1H), 7.88 (dd, 1H, J =
3.4 Hz, J = 5.6 Hz), 7.75 (m, 2H), 5.74 (ddt, 1H, Jd1 = 17.1 Hz,
Jd2 = 10.5 Hz, Jt = 5.6 Hz), 5.41 (br s, 1H), 5.21 (d, 1H, J =
17.1 Hz), 5.11 (d, 1H, J = 10.2 Hz), 3.78 (t, 2H, J = 6.0 Hz).
(S)-N1 -Allyl-N2 -Boc-N1 -(o-nitrobenzenesulfonyl)-5-(triisopro-
pylsilyloxy)pentane-1,2-diamine (L-8). To a stirred mixture of
alcohol L-7 (6.40 g, 17 mmol), triphenylphosphine (5.36 g, 1.2 eq.)
and N-allyl(o-nitrobenzene)sulfonamide (8.26 g, 2 eq.) in toluene–
◦
THF (7 : 1, 40 ml) at 0 C was added dropwise DIAD solution
1.9 M in toluene (11.7 ml, 1.3 eq.). The reaction was warmed up to
rt and stirred for 3 h. The reaction was concentrated and purified
by flash chromatography using hexane–EtOAc (90 : 10 to 70 : 30)
to furnish product L-8 (6.25 g, 61%). 1H NMR (CDCl3) d 8.03 (d,
1H, J = 6.8 Hz), 7.65 (m, 3H), 5.61 (m, 1H), 5.25 (d, 1H, J =
17.0 Hz), 5.16 (d, 1H, J = 10.2 Hz), 4.62 (d, 1H, J = 9.0 Hz), 4.09
(m, 1H), 3.92 (dd, 1H, J = 7.5 Hz, J = 16.3 Hz), 3.83 (m, 1H),
3.68 (m, 2H), 3.45 (dd, 1H, J = 10.2 Hz, J = 14.4 Hz), 3.21 (dd,
1H, J = 4.9 Hz, 14.6 Hz), 1.61 (m, 3H), 1.41 (m, 10H), 1.05 (m,
21H). 13C (CDCl3) d 155.8, 147.9, 134.0, 133.4, 132.0, 131.7, 130.9,
124.1, 119.9, 79.2, 62.8, 50.4, 49.6, 47.6, 29.3, 29.1, 28.3, 18.0, 12.0.
FTIR (cm−1) 2942, 2866, 1707, 1545, 1509, 1454, 1365, 1244, 1163.
MS (FAB+) m/z 600 (M + H+); HRMS calcd f◦or C28H50N3O7SSi+
1
allylamino]-5-(triisopropylsilyloxy)pentanoate (4.34 g, 82%). H
NMR (CDCl3) d 8.06 (m, 1H), 7.67 (m, 2H), 7.59 (m, 1H), 5.93
(ddt, 1H, Jt = 6.8 Hz, Jd1 = 10.2 Hz, Jd2 = 17.1 Hz), 5.21 (dd,
1H, J = 1.2 Hz, J = 17.1 Hz), 5.10 (d, 1H, J = 10.2 Hz), 4.66
(dd, 1H, J = 5.4 Hz, J = 10.0 Hz), 4.10 (dd, 1H, J = 5.9 Hz, J =
16.3 Hz), 3.92 (dd, 1H, J = 7.2 Hz, J = 16.3 Hz), 3.71 (t, 2H, J =
5.7 Hz), 3.57 (s, 3H), 2.14 (m, 1H), 1.83 (m, 1H), 1.66 (m, 2H), 1.05
(m, 21H). 13C (CDCl3) d 171.4, 148.4, 134.9, 133.4, 131.4, 131.2,
124.0, 118.0, 62.3, 60.4, 52.1, 48.8, 29.5, 26.5, 18.0, 11.9. FTIR
(cm−1) 2943, 2865, 1743, 1544, 1461, 1437, 1354, 1164, 1101. MS
(FAB+) m/z 529 (M + H+); HRMS calcd for C24H41N2O7SSi+
(MH+) 529.2398, found 529.2396. The allylnosylamide (4.34 g,
8.21 mmol) was dissolved in DMF (95 ml) and was treated
with mercaptoacetic acid (2.8 ml, 5 eq.) and LiOH·H2O (3.40 g,
9.85 eq.). The reaction was stirred for an hour at rt. The reaction
was diluted in EtOAc and NaHCO3. Aqueous layer was extracted
twice with EtOAc. Combined organic layers were washed with
brine, dried over MgSO4 and concentrated. The oil was purified
by flash chromatography using hexane–EtOAc (80 : 20) to furnish
L-10 (2.55 g, 90%, 63% from L-6). 1H NMR (CDCl3) d 5.85 (ddt,
1H, Jd1 = 16.8 Hz, Jd2 = 10.2 Hz, Jt = 6.1 Hz), 5.17 (dt, 1H, Jd =
17.3 Hz, Jt = 1.5 Hz), 5.08 (dt, 1H, Jd = 10.2 Hz, Jt = 1.5 Hz),
3.72 (s, 3H), 3.68 (t, 2H, J = 6.0 Hz), 3.31–3.24 (m, 2H), 3.11
(ddd, 1H, J = 1.2 Hz, J = 6.1 Hz, J = 13.7 Hz), 1.79 (br s, 1H),
1.72 (m, 2H), 1.59 (m, 2H), 1.05 (m, 21H). 13C (CDCl3) d 175.9,
136.3, 116.2, 62.9, 60.4, 51.6, 50.7, 29.9, 29.0, 18.0, 11.9. FTIR
(cm−1) 2943, 2865, 1737, 1462, 1196, 1171, 1103. MS (FAB+) m/z
344 (M + H+); HRMS calcd for C18H38NO3Si+ (MH+) 344.2615,
found 344.2620.
21
(MH+) 600.3133, found 600.3146. [a]D −22.3 (c = 1.04).
Methyl (S)-2-(N-allylamino)-5-(triisopropylsilyloxy)pentanoate
(L-10) [allyl-Hnv(TIPS)-OMe]. Method 1: L-6 (500 mg,
1.24 mmol) was dissolved in DCM (9 ml) and cooled down to
0
◦C. TFA (8 ml) was added dropwise and the reaction was
stirred for an hour at 0 ◦C. The reaction was diluted with toluene
and was concentrated. The oil was diluted again in toluene and
concentrated to give the TFA salt, which was used without further
˚
purification. Dried, powdered 4 A molecular sieves (2.5 g) were
suspended in DMF (13 ml). LiOH (100 mg, 2.13 eq.) was added
and the mixture was it stirred for 20 min. Amine was added and
the reaction was stirred for 45 additional min. Allyl bromide
(100 ll, 1.0 eq.) was finally added and the reaction was stirred
overnight. The reaction was filtered, diluted in EtOAc and washed
3 times with water. The combined aqueous layers were extracted
twice with EtOAc. The combined organic layers were washed with
brine, dried over MgSO4 and concentrated. The oil was purified
by flash chromatography using DCM–EtOAc (90 : 10) to furnish
L-10 (246 mg, 64%).
Method 2: the 3 : 1 mixture of L-6 L-6 and TIPSOH (5.5 g,
13.6 mmol of L-6) was dissolved in DCM (70 ml) and cooled
down to 0 ◦C. TFA (60 ml) was added dropwise and the reaction
was stirred for an hour at 0 ◦C. The reaction was diluted with
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Methyl (S)-2-[N-allyl-3-(4-benzyloxyphenyl)propamido]-5-(tri-
isopropylsilyloxy)pentanoate (13a). Amine L-10 (1.97 g,
5.73 mmol), 3-(4-benzyloxyphenyl)propanoic acid 11 (2.94 g,
2 eq.) and HATU (4.36 g, 2 eq.) were dissolved in DMF (55 ml).
Et3N (2.4 ml, 3 eq.) was added dropwise and the reaction was
stirred for 3 h at rt. The reaction was partitioned between ether
and HCl 0.5M solution. The aqueous phase was extracted twice
with ether. The combined organic phases were successively washed
with water and brine, dried over MgSO4 and concentrated. The
oil was purified by flash chromatography using hexane–EtOAc
(90 : 10 to 80 : 20) to furnish 13a (3.18 g, 95%). 1H NMR (CDCl3)
d 7.43–7.26 (m, 5H), 7.12 (m, 2H), 6.89 (m, 2H), 5.78 (m, 1H),
5.19–5.13 (m, 2H), 5.03 (s, 2H), 4.87 (dd, 1H, J = 6.1 Hz, J =
9.0 Hz), 3.95 (dd, 1H, J = 5.6 Hz, J = 17.6 Hz), 3.81 (dd, 1H, J =
5.4 Hz, J = 17.8 Hz), 3.68 (m, 5H), 2.92 (m, 2H), 2.62 (dd, 1H,
J = 6.6 Hz, J = 8.8 Hz), 2.06 (m, 1H), 1.82 (m, 1H), 1.53 (m, 2H),
1.06 (m, 21H). 13C (CDCl3) d 173.4, 172.1, 157.2, 137.1, 134.1,
133.6, 132.9, 129.3, 128.6, 127.9, 127.4, 117.2, 114.8, 70.0, 62.7,
57.3, 52.0, 48.8, 35.6, 30.4, 29.8, 25.6, 18.0, 11.9. FTIR (cm−1)
2943, 2865, 1738, 1649, 1611, 1510, 1455, 1238, 1175, 1105.
with HCl 0.5 M. The combined aqueous phases were reextracted
twice with ether. The combined organic phases were washed with
water, brine, dried over MgSO4 and concentrated. Purification by
flash chromatography using hexane–EtOAc (75 : 25) furnished
(14 + 15) (3.24 g, 88%) as a 1 : 1 mixture of two diastereomers. MS
(FAB+) m/z 1049 (M + H+), 1071 (M + Na+); HRMS calcd for
C56H89N4O9SSi2 (MH+) 1049.5889, found 1049.5879. Analytical
+
HPLC on CHIRACEL OD–H 0.46 × 25 cm in hexane–iPrOH
(90 : 10 to 5 : 95 in 30 min) give rt1 = 9.27 min and rt2 =
10.61 min.
(2R)- and (2S)-2-[N-Allyl-(4-benzyloxyphenyl)acetamido]-N-
[(2S)-1-(N-allyl-o-nitrophenylsulfonamido)-5-triisopropylsilyloxy-
pentan-2-yl]-5-triisopropylsilyloxypentanamide [(2S,2ꢀS)-18 and
(2R,2ꢀS)-19]. Acid 3b (1.38 g, 2.49 mmol) and amine L-4 (1.62 g,
1.3 eq.) were coupled as described for the synthesis of 14/15.
Purification by flash chromatography using hexane–EtOAc (75 :
25) furnished two diastereomers (2S,2ꢀS)-18 (1.11 g, 43%) and
1
(2R,2ꢀS)-19 (1.23 g, 48%). First to elute (2S,2ꢀS)-18: H NMR
(CDCl3) d (signal for major rotamer) 8.01 (m, 1H), 7.66 (m, 3H),
7.46–7.32 (m, 5H), 7.15 (d, 2H, J = 7.5 Hz), 6.93 (d, 2H, J =
7.5 Hz), 6.52 (br d, 1H, J = 9.0 Hz), 5.77 (m, 1H), 5.55 (m, 1H),
5.25–5.08 (m, 4H), 5.04 (s, 2H), 4.57 (br s, 1H), 4.13–3.87 (m, 4H),
3.65 (m, 7H), 3.45 (dd, 1H, J = 10.0 Hz, J = 14.9 Hz), 3.16 (dd,
1H, J = 4.9 Hz, J = 14.9 Hz), 2.01 (m, 1H), 1.89 (m, 1H), 1.48 (m,
6H), 1.05 (m, 42H). MS (FAB+) m/z 1035 (M + H+), 1057 (M +
Na+); HRMS calcd for C55H87N4O9Si2S+ (MH+) 1035.5727, found
1035.5726. Second to elute (2R,2ꢀS)-19: 1H NMR (CDCl3) d 7.96
(m, 1H), 7.68–7.60 (m, 3H), 7.43–7.31 (m, 5H), 7.17 (d, 2H, J =
8.5 Hz), 6.91 (d, 2H, J = 8.5 Hz), 6.28 (d, 1H, J = 9.8 Hz), 5.86
(m, 1H), 5.50 (m, 1H), 5.28 (m, 3H), 5.14 (d, 1H, J = 10.3 Hz),
5.07 (t, 1H, J = 7.8 Hz), 5.03 (s, 2H), 4.12 (m, 2H), 3.99 (dd, 1H,
J = 8.0 Hz, J = 16.3 Hz), 3.91 (dd, 1H, J = 4.6 Hz, J = 18.0 Hz),
3.78–3.60 (m, 7H), 3.44 (dd, 1H, J = 10.5 Hz, J = 14.4 Hz), 3.11
(dd, 1H, J = 4.1 Hz, J = 14.4 Hz), 2.01 (m, 1H), 1.74 (m, 1H),
1.50 (m, 6H), 1.04 (m, 42H). MS (FAB+) m/z 1035 (M + H+), 1057
(S)-2-[N-Allyl-3-(4-benzyloxyphenyl)propanamido]-5-(triisopropyl-
silyloxy)pentanoic acid (3a). Ester 13a (3.18 g, 5.46 mmol)
was dissolved in THF–H2O–MeOH (3 : 1 : 1, 55 ml), cooled
◦
down to 0 C and treated with LiOH·H2O (688 mg, 3 eq.). The
◦
reaction was stirred for 3 h at 0 C and was diluted with EtOAc
and 0.5 M HCl solution. The aqueous phase was extracted with
EtOAc. The combined organic phases were washed with brine,
dried over MgSO4 and concentrated. The oil was purified by
flash chromatography using hexane–EtOAc (90 : 10 to 60 : 40)
1
to furnish 3a (2.72 g, 88%). H NMR (CDCl3) d 7.43–7.29 (m,
5H), 7.10 (m, 2H), 6.89 (m, 2H), 5.78 (ddt, 1H, Jd1 = 17.1 Hz,
Jd2 = 10.2 Hz, Jt = 4.9 Hz), 5.18 (m, 2H), 5.02 (s, 2H), 4.64 (dd,
1H, J = 6.3 Hz, J = 8.5 Hz), 3.96 (dd, 1H, J = 5.6 Hz, J =
17.6 Hz), 3.80 (dd, 1H, J = 5.4 Hz, J = 17.6 Hz), 3.69 (m, 2H),
2.92 (m, 2H), 2.63 (dd, 1H, J = 7.3 Hz, J = 9.0 Hz), 2.12 (m,
1H), 1.87 (m, 1H), 1.56 (m, 2H), 1.05 (m, 21H). 13C (CDCl3) d
175.7, 174.2, 157.2, 137.1, 134.1, 133.6, 132.9, 129.3, 128.6, 127.9,
127.4, 117.2, 114.8, 69.9, 62.6, 59.0, 50.0, 35.7, 30.4, 29.8, 25.5,
18.0, 11.9. FTIR (cm−1) 2942, 2865, 1731, 1649, 1610, 1545, 1510,
1462, 1239, 1176, 1105. MS (FAB+) m/z 568 (M + H+); HRMS
calcd for C33H50NO5Si+ (MH+) 568.3453, found 568.3454.
(M + Na+); HRMS calcd for C55H87N4O9SSi2 (MH+) 1035.5732,
+
found 1035.5743.
(2S,5S,6E)-2,5-Bis[3-(triisopropylsilyloxy)propyl]-1-[3-(4-ben-
zyloxyphenyl)propionyl]-7-(o-nitrobenzenesulfonyl)-1,4,7-triazacy-
cloundec-9-en-3-one [(2S,5S,6E)-22] and its (2R,5S,6E)-isomer
(23). The diastereomeric mixture 14/15 (3.2 g, 3.05 mmol) was
dissolved in DCM (500 ml, 6 mM). The solution was degassed
5 min by bubbling argon and Grubb’s I catalyst (250 mg, 0.1 eq.)
was added. The reaction was stirred for 12 h under reflux. The
catalyst (250 mg, 0.1 eq.) was added again and the reaction was
stirred for 12 h under reflux. Volatile compounds were removed
and the products were purified by flash chromatography using
hexane–EtOAc (70 : 30 to 60 : 40) and furnished two diastereomers,
(2S,5S,6E)-22 (0.406 g, 13%) and (2R,5S,6E)-23 (0.752 g, 25%).
(2R,S)-2-[N-Allyl-3-(4-benzyloxyphenyl)propanamido]-N-[(2S)-
1-(N-allyl-o-nitrophenylsulfonamido)-5-triisopropylsilyloxypentan-
2-yl]-5-triisopropylsilyloxypentanamide [(2R,S,2ꢀS)-(14/15)]. L-8
(3.00◦g, 5 mmol) was dissolved in DCM (30 ml) and cooled down
to 0 C. TFA (20 ml) was then added dropwise and the reaction
◦
was stirred for an hour at 0 C. Toluene (30 ml) was added and
DCM and TFA were removed under reduced pressure. Toluene
solution was diluted with EtOAc and saturated NaHCO3 solution
was carefully added. Aqueous phase was extracted twice with
EtOAc. Combined organic phases were washed with brine, dried
over MgSO4 and concentrated as an oil. L-4 (2.40 g, 96%) was
used without further purification. Acid 3a (2.00 g, 3.50 mmol),
amine L-4 (2.29 g, 1.3 eq.), HOAt (0.815 g, 1.70 eq.) and HATU
(2.68 g, 2.00 eq.) were dissolved in DMF (35 ml) and DIEA
(1.23 ml, 2.00 eq.) was added dropwise. The reaction was stirred
overnight at room temperature. The reaction was partitioned
between ether and HCl 0.5 M. The ether phase was washed again
1
First to elute (2S,5S,6E)-22 : H NMR (CDCl3) d 7.91 (d, 1H,
J = 7.6 Hz), 7.71–7.60 (m, 3H), 7.44–7.29 (m, 5H), 7.13 (d, 2H,
J = 8.3 Hz), 6.91 (d, 2H, J = 8.5 Hz), 6.21 (d, 1H, J = 5.9 Hz),
5.57 (d, 1H, J = 16.1 Hz), 5.45 (m, 1H), 5.13 (t, 1H, J = 7.8 Hz),
5.05 (s, 2H), 4.30 (d, 1H, J = 11.5 Hz), 3.92 (d, 1H, J = 9.3 Hz),
3.76 (dd, 1H, J = 5.1 Hz, J = 17.8 Hz), 3.68–3.51 (m, 5H), 3.29
(m, 2H), 3.14 (dd, 1H, J = 9.8 Hz, J = 12.9 Hz), 2.96 (t, 2H,
J = 8.5 Hz), 2.65 (t, 2H, J = 8.5 Hz), 2.00 (m, 1H), 1.77 (m,
1H), 1.40 (m, 6H), 1.05 (m, 42H). MS (FAB+) m/z 1021 (M+),
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977 (M+ − iPr). Second to elute (2R,5S,6E)-23 : 1H NMR (CDCl3)
d 7.95 (dd, 1H, J = 7.5 Hz, J = 1.7 Hz), 7.71–7.56 (m, 3H),
7.41–7.30 (m, 5H), 7.15 (d, 2H, J = 8.5 Hz), 6.91 (d, 2H, J =
8.5 Hz), 5.58 (d, 1H, J = 8.0 Hz), 5.50 (dt, 1H, Jd = 15.8 Hz,
Jt = 8.0 Hz), 5.36 (dd, 1H, J = 15.4 Hz, J = 5.1 Hz), 5.20 (t,
1H, J = 7.7 Hz), 5.01 (s, 2H), 3.92 (m, 2H), 3.71–3.63 (m, 7H),
3.54 (d, 1H, J = 4.9 Hz), 3.42 (d, 1H, J = 14.1 Hz), 3.05 (m, 2H),
2.93 (m, 1H), 2.67 (t, 2H, J = 7.5 Hz), 1.85 (m, 1H), 1.72 (m,
2H), 1.48 (m, 5H), 1.05 (m, 42H). MS (FAB+) m/z 1021 (M+), 977
(M+ − iPr).
(M + H+); HRMS calcd for C42H50N3O6+ (MH+) 692.3700, found
692.3712.
(2S,5S,6E)-2,5-Bis[(3-guanidino)propyl]-1-[3-(4-hydroxyphenyl)-
propionyl]-7-(2-naphthylacetyl)-1,4,7-triazacycloundec-9-en-3-one
[(2S,5S,6E)-54]. (2S,5S,6E)-38 (150 mg, 0.22 mmol), PPh3
(171 mg, 3 eq.) and N,Nꢀ-bis-Boc-guanidine (337 mg, 6 eq.) were
added in a round-bottom flask and submitted to a vacuum/argon
(3×). The products were dissolved in THF (3 ml) and the
reaction was cooled down to 0 ◦C. DIAD solution (1.9M, 450 ll,
3.9 eq.) was added dropwise and the reaction was stirred for
24 h at rt. The reaction was partitioned between water and
EtOAc. The organic phase was washed with brine, dried over
MgSO4 and concentrated. Purification by flash chromatography
using hexane–EtOAc (70 : 30 to 30 : 70) furnished 46 (approx.
25 mg by NMR) as an inseparable mixture with Ph3PO and
mono-guanidylated product (166 mg). The mono-guanidylated
product was resubmitted to Mitsunobu conditions and furnished
22 (approx. 47 mg by NMR) as an inseparable mixture with
Ph3PO. Mixtures of product 46 and Ph3PO were dissolved in
DCM (1.5 ml), treated with anisole (23 eq. based on approx.
calculated mass) and pentamethylbenzene (34 eq.). TFA (4.5 ml)
was added dropwise and the reaction was stirred for 8 h. The
solvent was evaporated and oil was partitioned between water
and ether. The water phase was lyophilized to give a yellowish
foam which was purified by RP-HPLC in water–acetonitrile (90 :
10 to 40 : 60 in 33 min) to give (2S,5S,6E)-54 (13 mg, 9%). MS
(FAB+) m/z 684 (M + H+); HRMS calcd for C37H50N9O4+ (MH+)
(2S,5S,6E)-2,5-Bis[3-(triisopropylsilyloxy)propyl]-1-[3-(4-benzyl-
oxyphenyl)propionyl]-7-(2-naphthylacetyl)-1,4,7-triazacycloundec-
9-en-3-one [(2S,5S,6E)-30]. (2S,5S,6E)-22 (612 mg, 0.6 mmol)
was dissolved in DMF (6 ml) and was treated with mercaptoacetic
acid (0.25 ml, 6 eq.) and LiOH·H2O (280 mg, 11 eq.). The
reaction was stirred for an hour at rt. The reaction was diluted
in EtOAc and NaHCO3. The aqueous layer was extracted twice
with EtOAc. The combined organic layers were washed with
brine, dried over MgSO4 and concentrated. The product was used
without further purification. It was dissolved in DMF (6 ml) and
naphthylacetic acid (223 mg, 2 eq.) and HATU (456 mg, 2 eq.)
were added. Et3N (0.25 ml, 3 eq.) was then added dropwise and
the reaction was stirred overnight at rt. Reaction was partitioned
between ether and sat. NaHCO3 solution. Aqueous phase was
extracted 3× with ether. The combined organic phases were
washed with water, brine, dried over MgSO4 and concentrated.
Purification by flash chromatography using hexane–EtOAc (70 :
1
23
30 to 65 : 35) furnished (2S,5S,6E)-30 (382 mg, 63%): H NMR
684.3986, found 684.3984. [a]D −51 (c = 0.1, AcOH).
(CDCl3) d for major rotamer 7.79 (m, 3H), 7.64 (s, 1H), 7.46–7.29
(m, 8H), 7.03 (d, 2H, J = 8.5 Hz), 6.89 (d, 2H, J = 8.7 Hz), 5.93
(d, 1H, J = 6.3 Hz), 5.43 (br d, 1H, J = 19.0 Hz), 5.03 (m, 3H),
4.31 (d, 1H, J = 13.7 Hz), 3.92–3.65 (m, 9H), 3.43–3.32 (m, 3H),
3.23 (dd, 1H, J = 10.7 Hz, J = 14.9 Hz), 2.79 (m, 2H), 2.48 (m,
1H), 2.28 (m, 1H), 1.98 (m, 1H), 1.73 (m, 1H), 1.54 (m, 3H), 1.36
(m, 3H), 1.03 (m, 42H). MS (FAB+) m/z 1004 (M + H+), 960
Biological testing. Stable CHO cell transfectants expressing
the CXCR4 variant were prepared as described previously.35 CHO
transfectants were harvested by treatment with citrate saline, and
then washed in cold binding buffer (PBS containing 2 mg ml−1
BSA). For ligand binding, the cells were resuspended in binding
buffer at 1 × 107 cells ml−1, and 100 lL aliquots were incubated
with 0.1 nM of [125I]-SDF-1 (Perkin-Elmer Life Sciences) for 1 h on
ice under constant agitation. Free and bound radioactivities were
separated by centrifugation of the cells through an oil cushion,
and bound radioactivity was measured with a gamma-counter
(Cobra, Packard, Downers Grove, IL). The inhibitory activity of
test compounds was determined based on the inhibition of [125I]-
SDF-1 binding to CXCR4 transfectants (IC50).
(M+ − i-Pr); HRMS calcd for C60H90N3O6Si2 (MH+) 1004.6368,
+
found 1004.6376.
(2S,5S,6E)-2,5-Bis(3-hydroxypropyl)-1-[3-(4-benzyloxyphenyl)-
propionyl]-7-(2-naphthylacetyl)-1,4,7-triazacycloundec-9-en-3-one
[(2S,5S,6E)-38]. (2S,5S,6E)-30 (383 mg, 0.38 mmol) was dis-
solved in THF (3 ml) and was cooled down to −20 ◦C. TBAF
solution (1M, 2 ml, 4 eq.) was added and the reaction was stirred
for 48 h. TBAF solution (1M, 1 ml, 2 eq.) was added again and
the reaction was stirred for 48 additional hours. The reaction was
diluted with EtOAc and was quenched with water. The organic
phase was washed twice with water. The combined aqueous phases
were extracted 3× with EtOAc–hexane (9 : 1). The combined
organic phases were washed with brine, dried over MgSO4 and
concentrated. Purification by flash chromatography using hexane–
EtOAc (50 : 50), hexane–EtOAc–MeOH (48 : 48 : 4) and EtOAc–
MeOH (92 :◦8) furnished (2S,5S,6E)-38 (206 mg, 78%): 1H NMR
(CDCl3, 40 C) d 7.78 (m, 3H), 7.59 (s, 1H), 7.45–7.29 (m, 8H),
7.00 (d, 2H, J = 8.8 Hz), 6.87 (d, 2H, J = 8.5 Hz), 5.47 (m,
1H), 5.07 (m, 1H), 5.01 (s, 2H), 4.26 (d, 1H, J = 13.7 Hz), 3.83
(s, 2H), 3.77 (m, 2H), 3.61–3.50 (m, 5H), 3.31 (m, 3H), 3.20 (dd,
1H, J = 10.7 Hz, J = 14.1 Hz), 2.75 (m, 2H), 2.45 (m, 1H), 2.23
(m, 1H), 1.87 (m, 1H), 1.69–1.40 (m, 7H). MS (FAB+) m/z 692
References
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