Applications of 3-aminolactams: Design, synthesis, and biological evaluation of a library of potential dimerisation inhibitors of HIV1-protease

Article abstract of DOI:10.1039/c2ob25291k

In the context of our studies on the applications of 3-aminolactams as conformationally restricted pseudodipeptides, we report here the synthesis of a library of potential dimerisation inhibitors of HIV1-protease. Two of the pseudopeptides were active on the wild type virus (HIV1) at micromolar levels (EC₅₀). Although the peptides showed lower anti-viral activity than previously reported dimerisation inhibitors, our results demonstrate that the piperidone moiety does not prevent cell penetration, and hence that such derivatization is compatible with potential anti-HIV treatment.

Full text of DOI:10.1039/c2ob25291k

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PAPER
Applications of 3-aminolactams: design, synthesis, and biological evaluation
of a library of potential dimerisation inhibitors of HIV1-protease†
Eulàlia Pinyol,a Silvia Frutos,a Dolors Grillo-Bosch,a Ernest Giralt,a,b Bonaventura Clotet,c Jose A. Estéc
and Anna Diez*a,d
Received 9th February 2012, Accepted 5th April 2012
DOI: 10.1039/c2ob25291k
In the context of our studies on the applications of 3-aminolactams as conformationally restricted
pseudodipeptides, we report here the synthesis of a library of potential dimerisation inhibitors of HIV1-
protease. Two of the pseudopeptides were active on the wild type virus (HIV1) at micromolar levels
(EC50). Although the peptides showed lower anti-viral activity than previously reported dimerisation
inhibitors, our results demonstrate that the piperidone moiety does not prevent cell penetration, and hence
that such derivatization is compatible with potential anti-HIV treatment.
Introduction
Drugs currently used as inhibitors of HIV1 protease (HIV1-PR),
such as ritonavir and indinavir, interact directly with the active
site of the enzyme.1 However, since the demonstration that the
active form of HIV1-PR is dimeric,2,3 dimer assembly has been
seen as an alternative inhibitor target.3,4 Molecules of the
99-residue HIV1-PR monomer dimerize by interdigitation of the
five amino acids at the N- and C-termini (Fig. 1).
Meek et al. first proposed that small molecules able to comp-
lement these sequences could cap the monomers and would thus
Fig. 1 HIV1 protease and its dimerisation interface.
be good drug candidates (Fig. 2).3 The validity of this approach
was supported by Zhang et al., who reported peptide-induced
dissociation of HIV1-PR dimers (Fig. 2),5 and confirmed by the
observation that terminal peptides inhibit the protease by binding
to its dimerisation interface.6
With the aim of strengthening interaction with the protease
monomer, Schramm, Reboud-Ravaux et al. investigated
dimerisation inhibition of HIV1-PR by lipopeptides,7 and by
“tweezer” compounds in which the peptide chains are separated
by the charged heterocycles guanidine8 and quinoline.9 In paral-
lel, Chmielewski et al. made a systematic study of the structure–
Fig. 2 Inhibition of the dimerisation.5
activity relationships of compounds formed by peptide chains
separated by more flexible linkers.10,11 The structure–activity
relationships in the field of HIV1-PR dimerisation inhibitors
were extensively reviewed by Camarasa et al.12 In general, the
inhibitory activity of the reported compounds has been tested
in vitro and best activities are in the range of Kid = 150–400 nM.
More recently, Bannwarth et al. have reported the activity of a
series of alkyltripeptides including the potent dimerisation
inhibitor palmitoyl-Leu-Glu-Tyr (Kid = 0.3 nM).13
aInstitute for Research in Biomedicine, Barcelona Science Park,
08028-Barcelona, Spain
bDepartament de Química Orgànica, Facultat de Química, Universitat
de Barcelona, 08028-Barcelona, Spain
cFundació IrsiCaixa, Hospital Universitari Trias i Pujol, Badalona,
Spain
dLaboratori de Química Orgànica, Facultat de Farmàcia, Universitat de
Barcelona, 08028-Barcelona, Spain. E-mail: adiez@ub.edu
†Electronic supplementary information (ESI) available: NMR spectra of
compounds 3a and 3b, HPLC and MS of compounds 4–18cc, and
results on cultured cells are provided. See DOI: 10.1039/c2ob25291k
In the context of our studies on applications of conformation-
ally restricted lactam dipeptides,14 we report here the design and
synthesis of a collection of small molecules that potentially act
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the structures and the chemical yields, as well as inhibition
activity (EC50). Compound 7 had already been reported (IC50
=
1.1 μM),19 and we prepared it as a reference for our studies.
Compounds 4 and 5 correspond to the terminal portions of the
HIV1-PR, and had shown some activity in early studies.6b Com-
pounds 6cc, 12cc, 14cc, 16cc, 17cc and 18cc were obtained in
<5% yield, as the cross-coupling products (indicated as “cc” in
Scheme 1).
Fig. 3 Lactams used for the synthesis of the library.
Anti-HIV activity in cell culture20 (EC50)
All compounds were tested for activity in cultured cells infected
with wild type HIV1 virus. The four most potent compounds
were then tested on cells infected with the multidrug resistant
HIV1 strain IRLL98DPRO.21 The test was based on the MTT
colorimetric method that measures cellular proliferation,22
specifically applied to HIV1-PR inhibitors.23 AZT, AMD3100,
ritonavir and indinavir were used as the references. The concen-
trations at which these compounds were 50% effective (EC50)
were non-toxic to the cells (CC50 was also determined).24
The results showed that compounds 14 and 15 are active
against wild type HIV1 in the μM range. HIV1-PR inhibitors
indinavir and ritonavir are active at the nM scale. Although com-
pounds 14 and 15 do not show strong anti-viral activity, our
results show that the presence of lactam moieties 1 and 2 in their
structures does not prevent their entry into cells. Compounds 14
and 15 showed no activity against the highly resistant mutant. In
comparison with other dimerisation inhibitors reported, such as
those based on a bicyclic guanidinium subunit linked to short
peptide mimics of the terminal protease sequences8 or alkyl-
tripeptides,13 again, compounds 14 and 15 are less potent.
by capping the monomers of HIV1-PR, thus preventing
dimerisation and interrupting the virus life cycle. We have paid
particular attention to balancing hydrophilicity and hydrophobi-
city, and to keeping molecular weight low, in order to facilitate
permeation of the inhibitors through the cell membrane. Our
design is based on the use of 3-aminolactams 1 and 2 (Fig. 3) as
turn inducers. Compound 1 was first reported by Freidinger et al.
as a turn inducer when inserted in a peptide chain,15 and it has
been used in our laboratory to build a library of potential tryptase
inhibitors.14a Compound 2 is a beta-turn mimetic by itself.14d
Compounds 1 and 2 share the ability to act as both acceptors and
donors in β-strand like structures. We find that two of our com-
pounds enter cells efficiently, and so open a new avenue for
development of anti-HIV1-PR drugs.
Results and discussion
Potential dimerisation inhibitors design and synthesis
On the basis of the published data, we designed a library of com-
pounds by combining short peptide chains, a flexible carbon
chain spacer, and lactam turn-inducers 114a and 214d (Fig. 3).
The first series of compounds consisted of short peptides and
of short peptides bearing a free flexible carbon chain (Fig. 4).
In the second series (Fig. 5), another peptide chain, parallel to
the first peptide, was bound to the spacer, so the compound
would interdigitate with the antiparallel terminal chain of the
HIV1-PR.
The synthesis of the first series of the library was performed
on solid phase, using a standard Fmoc/tBu protocol16 on Wang
resin for the acid-terminated compounds and on MBHA Rink
amide for the amide-terminated peptides (Scheme 1). Piperidine
was used to cleave the Fmoc group; a DIPCDI–HOBt mixture
was used as the coupling agent for the commercial amino acids;
PyBOP–HOBt in the presence of DIEA was used to couple the
lactam amino acids 1 and 2, and ninhydrin (Kaiser test)17 was
used to monitor chain elongation.
For the second series (Scheme 1), we prepared the additional
dipeptides separately, using the same protocol but in solution.
We then coupled the dipeptide to compounds of type I still
attached to the resin, using DIPCDI–HOBt, and monitored the
coupling using malachite green.18 Side-chain deprotection (when
needed) and release of the compounds were performed with
TFA–H2O (95 : 5). Compounds purified by HPLC were 85–95%
pure.
Conclusion
We have built a library of potential dimerisation inhibitors of
HIV1-PR and evaluated the activity of the compounds. We have
found that two compounds (14 and 15) are active in the low
micromolar range on cells infected with the wild type virus, and
are not cytotoxic at the active concentration. Compounds 14 and
15 contain the piperidone moiety, showing that its inclusion in
the structure does not prevent cell penetration.
Experimental section
Semipreparative HPLC was performed on a Waters Delta Prep
HPLC4000, with a dual wavelength detector (model 2487) and a
fraction collector. The semi-preparative column used was Sym-
metry C18, 19 × 100 mm, 5 μm (Waters). Analytical HPLC was
performed on a Waters analytical HPLC instrument including a
model binary pump (model 1525), an autoinjector (model 717
plus), and a dual wavelength detector model 2487. The analyti-
cal column used was Symmetry C18, 4.6 × 150 mm, 5 μm
(Waters). Mass spectra were determined by MALDI-TOF (Pro-
teomics Analyzer 4700, Applied Biosystems). Exact mass
spectra were recorded on a LTQ-FT Ultra (Thermo Scientific)
spectrometer. Samples were previously dissolved in 1 mL of
H2O–CH3CN–FA (1 : 1 : 1), and a 1 to 100 dilution in H2O–
CH3CN–FA (1 : 1 : 1) was used for the analysis. The UV-Vis
In this manner, 25 pure compounds were obtained in sufficient
quantity to allow biological evaluation. Fig. 4 and 5 summarize
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Fig. 4 Library of potential HIV1-PR inhibitors: first series.
3-Allyloxycarbonylamino-2-oxopiperidinyl-1-(phenylalanylamide)
acetamide (3a and 3b)
spectrometer was a Shimadzu (model UV-2501 PC). Compounds
were dried in a Freezamobile 12 EL (Virtis) lyophilizer. The cen-
trifuge was a Beckman Coulter, model Allegra 21. The amino
acid analyses were performed using a Beckman System 6300.
Resins, aminoacids and coupling reagents were purchased from
Novabiochem. Dry solvents were purchased from SDS or
Aldrich. All final compounds showed 95–98% HPLC purities.
To a solution of compound 112 (330 mg, 0.390 mmol) in
CH2Cl2 (13 mL), a mixture of DIEA (204 μL, 1.171 mmol),
PyBOP (304 mg, 0.585 mmol) and HOBt (896 mg,
0.585 mmol) was added. Then, a solution of H-Phe-NH2·HCl
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Fig. 5 Library of potential HIV1-PR inhibitors: second series.
(86 mg, 0.429 mmol) in CH2Cl2 (1 mL) with 2 drops of DIEA
was added. The mixture was stirred for 12 h, the solvent was
evaporated and the residue was purified by column chromato-
graphy (CH2Cl2). Semipreparative HPLC (H2O–MeCN, 75 : 25
to 68 : 32 gradient, 15 min) allowed separation of the diastereo-
mers. Compound 3a (172 mg, 33%): IR (NaCl): ν 3306, 3061,
3.18 (brs, 2H, H-βb, H-2a), 3.43 (brs, 1H, H-6′a), 3.55 (brs,
1H, H-6′b), 4.00 (brs, 1H, H-3′), 4.35 (brs, 1H, H-2b), 4.52 (brs,
2H, OCH2), 4.68 (brs, 1H, H-α), 5.19 (d, J = 8.8 Hz,
1H, CHvCH2), 5.27 (d, J = 16.8 Hz, 1H, CHvCH2),
5.79–6.10 (m, 2H, CHvCH2, NH), 6.74 (brs, 1H, NH),
7.18–7.37 (m, 5H, Ph). 13C-NMR (CDCl3, 100 MHz) δ 21.3
(C-5′), 28.0 (C-4′), 37.6 (C-β), 50.1 (C-6′), 52.2 (C-3′), 52.3
(C-2), 54.9 (C-α), 68.4 (OCH2), 118.2 (CHvCH2), 127.2
1
3023, 2940, 2870, 1666 cm−1. H-NMR (CDCl3, 400 MHz) δ
1.88 (brs, 2H, H-5′), 2.26 (brs, 2H, H-4′), 3.04 (brs, 1H, H-βa),
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36.6 (C-β), 50.1 (C-6′), 52.3 (C-3′), 53.0 (C-2), 54.6 (C-α), 66.5
(OCH2), 118.4 (CHvCH2), 126.9 (Ph-p), 128.7 and 129.4
(Ph-o,m), 132.4 (CHvCH2), 137.9 (Ph-ipso), 156.9 (CvO
carbamate), 168.2 (CvO), 170.6 (CvO), 174.5 (CvO).
MS-ESI (m/z) 425.2 (M + Na)+, 403.2 (M + H)+, 239.2
(M − Phe − NH2)+, 211.2 (M − CONH − Phe − NH2)+. Calcd
mass for C20H27N4O5 403.19760. Found: 403.19760. Calcd for
C20H26N4O5: C, 59.69%; H, 6.51%; N, 13.92%. Found: C,
59.65%; H, 6.21%; N, 13.86%.
General protocol for the solid phase peptide synthesis
The compounds in the library were obtained by solid phase syn-
thesis, using standard protocols13 on Wang (Novabiochem,
0.96 mmol g−1
) or Rink amide (Novabiochem MBHA,
0.66 mmol g−1) resins. Coupling of commercial amino acids
(Novabiochem), of dipeptides H-Asn-Phe-OtBu and Ile-Phe-
NH2, as well as of adipic and hexadecandioic acids (Aldrich)
were performed using DIPCDI (3 equivalents) and HOBt
(3 equivalents). Oxazolopiperidone 1 and lactam 2 were coupled
using PyBOP (3 equivalents), HOBt (3 equivalents), and DIEA
(6 equivalents). Incorporation of amino acids was monitorized
by the ninhydrin test,14 and that of diacids by malachite green
test.15 All compounds were purified (95 to 98% purity) by semi-
preparative HPLC (milli-Q H2O with 0.1% of TFA, MeCN with
0.1% of TFA; flux, 15 mL min−1; UV detection, λ = 220 and
254 nm). The chemical yields given correspond to the total
syntheses described, after final HPLC purification.
H2N-Ser(tBu)-Thr(tBu)-Leu-Asn(Trt)-Phe-OH
(compound 4, 327 mg, 21%)
The synthesis was done on a 62 μmol scale using Wang resin.
Purification conditions: 30 to 80% MeCN in 50 min. Analytical
HPLC: tR = 13.20 min (0 to 100% MeCN in 15 min);
tR = 11.428 min (30 to 80% MeCN in 15 min). AAA: Ser, 0.92
(1); Thr, 1.02 (1); Leu, 1.08 (1); Asn, 0.97 (1); Phe, 1.14 (1).
MALDI-TOF: 957.43 (M + Na)+, 979.42 (M + K)+. Calcd mass
for C53H71N6O9 935.52770. Found: 935.52814.
Scheme 1 Synthesis of the compounds.
HNPro-Gln-Ile-Thr-Leu-Trp-OH (compound 5, 505 mg, 53%)
(Ph-p), 128.9 and 129.0 (Ph-o,m), 132.6 (CHvCH2), 136.8 (Ph-
ipso), 156.8 (CvO carbamate), 169.2 (CvO), 171.2 (CvO),
175.0 (CvO). MS-ESI (m/z) 425.2 (M + Na)+, 403.2 (M + H)+,
239.2 (M − PheNH2)+, 211.2 (M − CONH − PheNH2)+. Calcd
mass for C20H27N4O5 403.19760. Found: 403.19753. Calcd for
C20H26N4O5: C, 59.69%; H, 6.51%; N, 13.92%. Found: C,
59.13%; H, 6.30%; N, 14.29%. Compound 3b (121 mg, 23%):
The synthesis was done on a 66 μmol scale using Wang resin.
Purification conditions: 30 to 90% MeCN in 30 min. Analytical
HPLC: tR = 9.53 min (0 to 100% MeCN in 15 min);
tR = 9.359 min (20 to 40% MeCN in 15 min). AAA: Pro, 0.90
(1); Glu, 1.04 (1); Ile, 0.89 (1); Thr, 1.00 (1); Leu, 1.06 (1); Trp,
0.92 (1). MALDI-TOF: 797.39 (M + Na)+, 795.37 (M + K)+.
Calcd mass for C37H57N8O9 757.42430. Found: 757.42363.
IR (NaCl) ν 3275, 3064, 3027, 2943, 2873, 1716, 1663 cm−1
.
1H-NMR (CDCl3, 400 MHz) δ 1.74–1.94 (m, 2H, H-5′),
2.08–2.24 (m, 2H, H-4′), 2.72 (brs, 2H, NH2), 3.02–3.12 (m,
3H, H-6′, H-βa), 3.24 (d, J = 16.0 Hz, 1H, H-2a), 3.43 (d,
J = 13.6 Hz, 1H, H-βb), 3.66–3.78 (m, 1H, H-3′), 4.53 (dd, J =
13.4 and 5.4 Hz, 1H, OCH2), 4.62 (dd, J = 13.4 and 5.0 Hz, 1H,
OCH2), 4.53–4.62 (m, 1H, H-2b), 4.74 (brs, 1H, H-α), 5.24 (d,
J = 10.4 Hz, 1H, CHvCH2), 5.33 (d, J = 16.8 Hz, 1H,
CHvCH2), 5.69 (brs, 1H, NH), 5.91 (ddd, J = 22.2, 10.8 and
5.4 Hz, 1H, CHvCH2), 6.58 (brs, 1H, NH), 7.18–7,44 (m, 5H,
Ph). 13C-NMR (CDCl3, 100 MHz): δ 21.5 (C-5′), 28.2 (C-4′),
[N-(15-Carboxypentadecanoyl)isoleucyl]phenylalanylamide
(compound 6, 7.2 mg, 19%)
The synthesis was done on a 66 μmol scale using Rink amide
resin. Purification conditions: 35 to 90% MeCN in 50 min.
Analytical HPLC: tR = 14.92 min (0 to 100% MeCN in 15 min);
tR = 14.87 min (30 to 80% MeCN in 15 min). MALDI-TOF:
568.4 (M + Na)+, 584.3 (M + K)+. Calcd mass for C31H52N3O5
546.39015. Found: 546.39019. Cross coupling product 6cc
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(2.1 mg, 4%). Analytical HPLC: tR = 15.21 min (0 to 100%
MeCN in 15 min); tR = 15.03 min (30 to 80% MeCN in
15 min). MALDI-TOF: 829.4 (M + Na)+, 845.4 (M + K)+.
Calcd mass for C46H73N6O6 805.38792. Found: 805.38815.
(M + Na)+, 859.3 (M + K)+. Calcd mass for C41H57N8O10
821.41922. Found: 821.41904.
N-(Oxazolipiperidonylisoleucinylphenylalanine)
hexadecanamide (compound 11, 12.5 mg, 36%)
N-Asparagylphenylalanyl N′-(isoleucylphenylalanylamide)
The synthesis was done on a 47 μmol scale using Rink amide
MBHA resin. Purification conditions: 40 to 90% MeCN in
50 min. Analytical HPLC: tR = 7.79 min (0 to 100% MeCN in
15 min); tR = 4.61 min (10 to 30% MeCN in 15 min). MALDI-
TOF: 750.4 (M + Na)+, 766.4 (M + K)+. Calcd mass for
C39H62N5O8 728. 45 929. Found: 728.45874.
hexadecanoyldiamide (compound 7, 7.5 mg, 14%)
The synthesis was done on a 66 μmol scale using Rink amide.
Purification conditions: 40 to 90% MeCN in 50 min. Analytical
HPLC: tR = 13.66 min (0 to 100% MeCN in 15 min);
tR = 12.56 min (30 to 80% MeCN in 15 min). MALDI-TOF:
829.5 (M + Na)+, 845.5 (M + K)+. Calcd mass for C44H67N6O8
807.50149. Found: 807.50173. The presence of the cross coup-
ling product 6cc was detected.
N-(Asparaginylphenylalanine) N′-(oxazolopiperidonyl-isoleucyl-
phenylalanylamide) hexanediamide (compounds 12 and 12cc)
The synthesis was done on a 99 μmol scale using Rink amide
resin. HPLC purification conditions: 20 to 70% MeCN in
45 min. Inhibitor 12 (35.3 mg, 42%). Analytical HPLC:
tR = 10.15 min (0 to 100% MeCN in 15 min); tR = 12.89 min
(20 to 40% MeCN in 15 min). MALDI-TOF: 871.4 (M + Na)+,
887.4 (M + K)+. Cross-coupling product 12cc (3.5 mg, 3%).
MALDI-TOF: 1051.5 (M + Na)+, 1067.5 (M + K)+.
N-(Isoleucylphenylalanylamide) 3-allyloxycarbonylamino-
2-oxopiperidin-1-acetamide (compounds 8a and 8b)
The synthesis was done on a 160 μmol scale using Rink amide
resin. HPLC purification and separation conditions: 27% MeCN
in 15 min. Compound 8a (23 mg, 28%). Analytical HPLC:
tR = 10.94 min (0 to 100% MeCN in 15 min); tR = 14.98 min
(20 to 40% MeCN in 15 min). MALDI-TOF: 538.2 (M + Na)+,
554.2 (M + K)+. Calcd mass for C26H38N5O6 516.28166.
Found: 516.28161. Compound 8b (25 mg, 30%). Analytical
HPLC: tR = 10.98 min (0 to 100% MeCN in 15 min);
tR = 15.36 min (20 to 40% MeCN in 15 min). MALDI-TOF:
538.3 (M + Na)+, 554.2 (M + K)+. Calcd mass for C26H38N5O6
516.28166. Found: 516.28162.
N-(Asparagylphenylalanine) 3-(allyloxycarbonylamino)-2-
oxopiperidin-1-acetamide (compound 13, 37 mg, 25%)
The synthesis was done on a 288 μmol scale using Wang resin.
HPLC purification conditions: 22% MeCN in 15 min. Analytical
HPLC: tR = 9.52 min (0 to 100% MeCN in 15 min);
tR = 9.12 min (20 to 40% MeCN in 15 min). MALDI-TOF:
540.2 (M + Na)+, 556.1 (M + K)+. Calcd mass for C24H32N5O8
518.22454. Found: 518.22425.
N-(Isoleucylphenylalanine) 3-carboxypentanoylamino-2-
oxopiperidin-1-acetamide (compounds 9 and 9cc)
The synthesis was done on a 198 μmol scale using Rink amide
resin. HPLC purification conditions: 23 to 24% MeCN in
15 min followed by 24 to 45% MeCN in 15 min. Inhibitor 9
(38 mg, 48%). Analytical HPLC: tR = 9.73 min (0 to 100%
MeCN in 15 min); tR = 10.08 min (20 to 40% MeCN
in 15 min). MALDI-TOF: 582.2 (M + Na)+, 598.2 (M + K)+.
Calcd mass for C28H42N5O7 560.30788. Found: 560.30747.
The cross-coupling product 9cc was detected: tR = 10.90 min
(0 to 100% MeCN in 15 min); tR = 16.41 min (20 to 40%
MeCN in 15 min). MALDI-TOF: 995.5 (M + Na)+, 1011.5
(M + K)+.
N-(Asparagylphenylalanine) 3-carboxypentanoylamino-2-
oxopiperidin-1-acetamide (compounds 14 and 14cc)
The synthesis was done on a 288 μmol scale using Wang resin.
HPLC purification conditions: 10 to 17% MeCN in 15 min.
Compound 14 (26 mg, 16%). Analytical HPLC: tR = 13.86 min
(0 to 100% MeCN in 15 min); tR = 14.76 min (30 to 80%
MeCN in 15 min). MALDI-TOF: 584.2 (M + Na)+, 600.2
(M + K)+. Cross-coupling product 14cc (7.4 mg, 3%):
tR = 9.28 min (0 to 100% MeCN in 15 min); tR = 8.47 min
(20 to 40% MeCN in 15 min). MALDI-TOF: 999.4 (M + Na)+,
1015.4 (M + K)+.
N-(Asparagylphenylalanine) N′-(oxazolopiperidonylisoleucyl-phenyl-
alanylamide) hexandiamide (compounds 10a and 10b)
N-(Isoleucylphenylalanylamide) N′-(aminopiperidonylglycyl-
asparagylphenylalanine)hexandiamide (compound 15, 80 mg, 34%)
The synthesis was done on a 205 μmol scale using Rink amide
resin. HPLC purification and separation conditions: 26% MeCN
in 15 min. Compound (3S)-10a (45 mg, 27%): analytical HPLC:
tR = 10.37 min (0 to 100% MeCN in 15 min); tR = 14.07 min
(26% MeCN in 15 min). MALDI-TOF: 871.4 (M + Na)+, 887.4
(M + K)+. Calcd mass for C41H57N8O10 821.41922. Found:
821.41923. Compound (3R)-10b (40 mg, 24%). Analytical
HPLC: tR = 10.48 min (0 to 100% MeCN in 15 min);
tR = 15.00 min (26% MeCN in 15 min). MALDI-TOF: 843.3
The synthesis was done on a 288 μmol scale using Wang resin.
HPLC purification conditions: 15 to 45% MeCN in 15 min,
followed by 45 to 100% in 5 min. Analytical HPLC:
tR = 13.88 min (0 to 100% MeCN in 15 min); tR = 14.80 min
(30 to 80% MeCN in 15 min). MALDI-TOF: 843.3 (M + Na)+,
883.3 (M + K)+. Calcd mass for C41H57N8O10 821.41922.
Found: 821.41931.
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W. Schramm, Antiviral Res., 1996, 30, 155.
7 (a) H. J. Schramm, E. De Rocín, M. Reboud-Ravaux, J. Buttner, A. Dick
and W. Schramm, Biol. Chem., 1999, 380, 593; (b) A. Bouras,
N. Bogetto, Z. Benatalah, E. De Rosny, S. Sicsic and M. Reboud-
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8 P. Breccia, N. Bogetto, R. Pérez-Fernández, M. Van Gool, M. Takahashi,
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The synthesis was done on a 288 μmol scale using Wang resin.
HPLC purification and separation conditions: 43% MeCN in
15 min. Compound 16 (49 mg, 18%): Analytical HPLC: tR
=
12.89 min (0 to 100% MeCN in 15 min); tR = 11.17 min (30 to
80% MeCN in 15 min). MALDI-TOF: 724.4 (M + Na)+, 740.4
(M + K)+. Calcd mass for C36H56N5O9 702.40726. Found:
702.40752. Cross coupling product 16cc (9.1 mg, 3%): Analyti-
cal HPLC: tR = 11.88 min (0 to 100% MeCN in 15 min); tR
=
9.61 min (30 to 80% MeCN in 15 min). MALDI-TOF: 1139.6
(M + Na)+, 1155.6 (M + K)+. Calcd mass for C56H81N10O14
1117.59337. Found: 1117.59344.
N-(Asparagylphenylalanine) 3-carboxypentandecanoylamino-
2-oxo-7,1-oxazolidin-9-carboxamide (compound 17, 47 mg, 22%)
9 N. Merabet, J. Dumonod, B. Collinet, L. Van Baelinghem, N. Bogetto,
S. Ongeri, F. Ressad, M. Reboud-Ravaux and S. Sicsic, J. Med. Chem.,
2004, 47, 6392.
The synthesis was done on a 288 μmol scale using Wang resin.
Purification conditions: 40 to 65% MeCN in 15 min. Analytical
HPLC: tR = 12.86 min (0 to 100% MeCN in 15 min); tR
=
10 (a) R. Zutshi, J. Franciskovich, M. Schultz, B. Schweitzer, P. Bishop,
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11 A dissociative inhibitory effect on HIV1-PR of non-peptide compounds,
such as marine didemnaketals A and B, and triterpenes has also been
reported: (a) X. Fan, G. R. Flentke and D. H. Rich, J. Am. Chem. Soc.,
1998, 120, 8893; (b) L. Quere, T. Wenger and H. J. Schramm, Biochem.
Biophys.Res. Commun., 1996, 227, 484.
11.21 min (30 to 80% MeCN in 15 min). MALDI-TOF: 752.2
(M + Na)+, 768.2 (M + K)+. Calcd mass for C37H56N5O10
730.40217. Found: 730.40232. The cross-coupling product 17cc
was also isolated (9%). Analytical HPLC: tR = 11.83 min (0 to
100% MeCN in 15 min); tR = 9.709 min (30 to 80% MeCN in
15 min). MALDI-TOF: 1195.63 (M + Na)+, 1211.64 (M + K)+.
Calcd mass for C58H81N10O16 1173.58320. Found: 1173.58323.
12 M.-J. Camarasa, S. Velázquez, A. San-Félix, M.-J. Pérez-Pérez and
F. Gago, Antiviral Res., 2006, 71, 260.
13 L. Bannwarth, T. Rose, L. Dufau, R. Vanderesse, J. Dumond, B. Jamart-
Grégoire, C. Pannecouque, E. De Clercq and M. Reboud-Ravaux, Bio-
chemistry, 2009, 48, 379.
N-(Isoleucylphenylalanylamide) N′-(oxazolopiperidonyl-aspara-
gylphenylalanine) hexandiamide (compounds 18 and 18cc)
The synthesis was done on a 288 μmol scale using Wang resin.
HPLC purification and separation conditions: 15 to 45% MeCN
14 (a) M. García, X. del Río, S. Silvestre, M. Rubiralta, E. Lozoya,
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9543; (c) P. Forns, J. Piro, C. Cuevas, M. Garcia, M. Rubiralta, E. Giralt
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65, 6992.
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R. Saperstein, Science, 1980, 210, 656; (b) R. M. Freidinger,
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in 15 min. Inhibitor 180 (40 mg, 16%): analytical HPLC: tR
=
10.23 min (0 to 100% MeCN in 15 min); tR = 13.40 min (20 to
40% MeCN in 15 min). MALDI-TOF: 871.3 (M + Na)+, 887.3
(M + K)+. Compound 18cc (11 mg, 4%): analytical HPLC: tR =
9.45 min (0 to 100% MeCN in 15 min); tR = 9.63 min (20 to
40% MeCN in 15 min). MALDI-TOF: 1055.4 (M + Na)+,
1071.3 (M + K)+. Calcd mass for C48H61N10O16 1033.42615.
Found: 1033.42580.
18 M. E. Attardi, G. Porcu and M. Taddei, Tetrahedron Lett., 2000, 41,
7391.
Acknowledgements
19 Y. S. Hwang and J. Chmielewski, J. Med. Chem., 2005, 48, 2239.
20 These tests were carried out at the IrsiCaixa AIDS Research Institute,
Hospital Universitari Germans Trias i Pujol, Badalona (Spain).
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This work has been supported by grants CTQ2004-1757/BQU,
CTQ2007-60764/BQU and SAF2010-21617-C02 (Ministerio de
Ciencia e Innovación, Spain), 2009SGR-1111 (Generalitat de
Catalunya). We also thank the Generalitat de Catalunya (XRB
and Grups Consolidats), and the Institute for Research in Biome-
dicine (IRB Barcelona-PCB) for the doctoral fellowship given to
E.P. We thank Dr David Lane for editorial assistance.
23 D. A. Davis, C. A. Brown, K. E. Singer, V. Wang, J. Kaufman,
S. J. Stahl, P. Wingfield, K. Maeda, S. Harada, K. Yoshimura,
P. Kosalaraksa, H. Mitsuya and R. Yarchoan, Antiviral Res., 2006, 72, 89.
24 CC50 is the concentration necessary for inducing 50% cell death. For all
compounds assayed CC50 was over 125 mM.
Notes and references
1 A. M. Wensing, N. M. van Maarseveen and M. Nijhuis, Antiviral Res.,
2010, 85, 59.
4354 | Org. Biomol. Chem., 2012, 10, 4348–4354
This journal is © The Royal Society of Chemistry 2012