Diastereoselective solution and multipin-based combinatorial array synthesis of a novel class of potent phosphinic metalloprotease inhibitors

Article abstract of DOI:10.1002/chem.200204456

The solution-phase synthesis and resolution of new phosphinopeptidic building blocks containing a triple bond was realized in high yields and optical purities (units 3a-d). The absolute configuration of the target compounds was unambiguously established b

Full text of DOI:10.1002/chem.200204456

FULL PAPER
Diastereoselective Solution and Multipin-Based Combinatorial Array
Synthesis of a Novel Class of Potent Phosphinic Metalloprotease Inhibitors
Anastasios Makaritis,[a] Dimitris Georgiadis,[a] Vincent Dive,[b] and Athanasios Yiotakis*[a]
Abstract: The solution-phase synthesis
and resolution of new phosphinopepti-
dic building blocks containing a triple
bond was realized in high yields and
optical purities (units 3a d). The abso-
lute configuration of the target com-
pounds was unambiguously established
by NMR studies. Apost-assembly diver-
sification strategy of these blocks was
developed through 1,3-dipolar cycload-
dition of a variety of in situ prepared
nitrile oxides. This strategy led to the
rapid and efficient diastereoselective
preparation of a novel class of isoxa-
zole-containing phosphinic peptides
(peptides 5a i). Solid-phase version of
this strategy was efficiently achieved on
multipin solid technology, by developing
a new protocol for the coupling of
P-unprotected dipeptidic blocks with
solid supported amino acids in a quanti-
tative and diastereoselective manner.
Optimization of dipolar cycloadditions
onto pin-embodied phosphinic peptides
allowed the convenient preparation of
this new class of pseudopeptides. The
crude phosphinic peptides (9a k) were
obtained in high yields and purity as
determined by RP-HPLC. Inhibition
assays of some of these peptides re-
vealed that they behave as very potent
inhibitors of MMPs, outmatching previ-
ously reported phosphinic peptides, in
terms of potency (Ki in the range of
few nm).
Keywords: combinatorial chemistry
¥ cycloaddition ¥ inhibitors ¥ metallo-
proteins ¥ solid-phase synthesis
Zn2+
Introduction
C terminus
N terminus
R1
O
O
P
Phosphinic peptides incorporating in their sequence the motif
-X1aaY[P(O)(OH)CH2]X2aa- are well recognized peptide
isosters and powerful inhibitors of many classes of enzymes,
especially the zinc-proteases (Figure 1).[1 Matrix metal-
N
H
N
H
O
R2
P1' position
P
1 position
3]
loproteases (MMPs),[4] bacterial collagenases,[2c, 5] enkephalin-
degrading enzymes,[6] other zinc metalloproteases,[1a, 7] HIV-
protease[8] and ligases[9] are a few examples of such enzymes
inhibited by this class of pseudopeptidic inhibitors.
Unlike hydroxamic, carboxylalkyl or thiol-containing in-
hibitors, which have been extensively developed to prepare
potent inhibitors of zinc-metalloproteases, phosphinic pep-
tides are devoid of toxicity, but more importantly these
compounds are stable in vivo,[3b, 10] allowing their use in vivo at
rather low concentration.[11a,b] Moreover, phosphinic peptide
chemistry offers the possibility to develop inhibitors able to
NH
O
O
O
H
N
O
P
O
NH2
Cbz
NH2
Ac
N
H
N
H
N
H
P
N
H
OH
O
OH
O
COOH
RXP407
RXP03
Figure 1. Standard representation of phosphinic peptides. P1 and P1'
positions are the segments of the peptide situated at the left and right-
hand site of the phosphinic moiety respectively (above). The chemical
structures of the two phosphinic inhibitors RXP407 and RXP03 (below).
[a] Prof. Dr. A. Yiotakis, A. Makaritis, Dr. D. Georgiadis
University of Athens, Department of Chemistry
Laboratory of Organic Chemistry
interact with both the prime and unprimed side of the active
site cleft, thus allowing optimization of the inhibitor selectiv-
ity by diversification of the P and P' positions of the inhibitor
(see for example RXP407 where the P2 residue is critical for
selectivity, Figure 1).[4b, 10, 12]
Recently, the phosphinic peptide RXP03[4b] (Figure 1),
bearing an unusual long side chain at the P1' position, was
reported as a highly potent inhibitor of different MMPs.[3b, 11a,c]
Panepistimiopolis Zografou 157 71, Athens (Greece)
Fax : (‡30)2107274498
E-mail: yiotakis@chem.uoa.gr
[b] Dr. V. Dive
¬
¬
CEA, Departement d×Ingenierie et
¬
d×Etudes des Proteines, CE-Saclay
91191 Gif/Yvette Cedex (France)
E-mail: vincent.dive@cea.fr
Chem. Eur. J. 2003, 9, 2079 2094
DOI: 10.1002/chem.200204456
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Yiotakis et al.
FULL PAPER
The resolution of the crystal structure of stromelysin-3
(MMP-11) complexed to RXP03[13] provided the basis for
the development of new phosphinic compounds with im-
proved properties in terms of selectivity and potency.
chain functionalized pseudopeptides, and especially peptides
comprising a heterocyclic core, since they have not yet been
reported. Taking into consideration the promising results
exhibited by RXP03 (see above), we chose to focus our
attention on the development of analogues of this inhibitor,
with particular emphasis on the P1' position. Indeed, it is well
known that this position is one of the key side chains for the
control of inhibitor potency and selectivity as far as MMPs are
concerned.[3b, 4b]
Regarding the nature of the heterocyclic core to be
incorporated in the peptides, the choice was not straightfor-
ward since no reports to similar compounds could be found.
Although many heterocyclic compounds have interesting
biological activities, an overview of the literature stimulated
our interest in the isoxazole ring. Isoxazole moieties (3,5-
disubsituted isoxazoles illustrated in Figure 2) have been
extensively utilized not only as precursors to versatile
intermediates for the synthesis of a variety of complex natural
products[17] but also as scaffolds for peptidomimetics,[18] and
core structures in medicinal chemistry, where they represent a
class of unique pharmacophores that are constituents of
diverse therapeutic agents.[19] Moreover, there exist several
naturally occurring isoxazoles with important pharmacolog-
ical activity (e.g. Muscimol, 4-hydroxyisoxazole, Figure 2).[20]
Despite the interesting properties of phosphinic peptides,
the difficulties often encountered in producing the appropri-
ate starting materials or lack of reactivity have greatly limited
the extensive use of this class of inhibitors.[9a, b, 14] Elongation
of phosphinic pseudodipeptidic units, which are obtained by a
Michael-type addition of silyl activated N-protected amino-
phosphinic acids to suitably substituted acrylates,[15] consti-
tutes the classical approach to prepare phosphinic (poly)pep-
tides.[4] Such an approach usually consists of several synthetic
steps and is absolutely dependent on the availability of the
starting materials. In addition, another drawback of this
strategy is that the chemical features of the P1 and P1'
positions (Figure 1) are inherently fixed, thus allowing
diversity introduction only in the positions framing the
dipeptidic block. Therefore, this is not a convenient strategy
to rapidly prepare large series of widely diversified phosphinic
peptide libraries.
Within the framework of our ongoing research concerning
the syntheses of novel polyfunctionalized phosphinic-based
inhibitors, we became interested in the development of
reliable and versatile alternative strategies that would provide
rapid access to the target molecules. Toward this end, we
speculated that the utilization of a modular strategy, which
would allow the desired diversification of a phosphinic
pseudopeptidic template at the very final step of the synthesis,
would be extremely useful. Such an approach is bound to give
a boost to the development of novel phosphinic inhibitors by
either parallel synthesis or combinatorial chemistry, as
preliminary studies from our laboratory have shown.[16]
Aliterature search revealed that most of the reported
phosphinic peptides generally involve amino acids such as
phenylalanine, alanine, glycine, valine that lack side-chain
functionality. However, we were interested in preparing side-
4
3
5
O
N
OH
H2N
H2N
HOOC
OH
I
OH
O
N
O
N
O
N
C
A
B
NHSO2
NH2
O
N
D
Figure 2. General structure of 3,5-disubstituted isoxazole core (top).
Examples of naturally occurring and synthetic isoxazoles of pharmaceutical
interest. A: Muscimol;[20] B: 4-hydroxyisoxazole;[20] C: ibotenic acid;[19d] D:
selective endothelin receptor A(ET A) antagonist.[19f]
Abstract in Greek:
Another key feature of the isoxazole ring that drew our
attention is its ionophoric properties. Generally, isoxazoles
coordinate to metals mainly through the nitrogen lone pair.[21]
Taking into account that our program is concerned with zinc-
metalloenzymes, which, in addition to zinc, may contain other
cations (e.g. Ca2‡), this isoxazole Lewis basicity may prove
extremely useful. Moreover, the enhanced hydrogen bonding
acceptor capability of the ring was envisioned to increase the
contact points of the inhibitors with the enzymes (Figure 3).
The above arguments render the isoxazole core a privileged
structure, which is well worthy of introduction into the field of
phosphinic pseudopeptides. Determined to design and devel-
op a novel approach to the synthesis of phosphinic peptides,
we wish to report herein our efforts toward this end. The
general structure of the target molecules is illustrated in
Figure 3. Useful precursors for the introduction of the
isoxazole ring into the P1' position of these peptides were
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Chem. Eur. J. 2003, 9, 2079 2094

Combinatorial Array Synthesis
2079 2094
obtained by Michael addition of silyl-activated versions of
aminophosphinic acids VI to acrylic derivatives VII. A n
alternative route might rely on the prior assembly of
isoxazole-containing dipeptide units derived from IV and
subsequent coupling with amino acids; nevertheless, such an
approach would involve more synthetic steps, and preliminary
experiments showed that it is less efficient, suffering from by-
product formation.
N
O
O
O
1,3-dipolar
cycloaddition
H
N
H
N
P
P
OH
OH
R1
O
O
R1
O
O
precursors
to isoxazole-containing
phosphinic inhibitors
isoxazole-containing
phosphinic inhibitors
The key-step in the strategy for the development of the
pseudopeptidic isoxazole-containing phosphinic inhibitors is
the construction of tripeptidic units II (Scheme 1) or 3
(Scheme 2).
S1' pocket of
enzyme
hydrogen bonds
?
?
N
O
Isoxazole
core
Zn2+
This synthesis commenced with the Michael addition of
silyl-activated a-amino phosphinic acids[15] to ethyl a-prop-
argylic acrylate.[23] Optically pure (R) Cbz N-protected amino
phosphinic acids[24] (l configuration) were chosen based on
enzymatic preferences, allowing thus the control of stereo-
chemistry at the P1 position of the final inhibitors. The use of
optically pure aminophosphinic acids had a great impact on
the stereochemistry of the newly generated stereogenic center
of the dipeptidic unit 1 (Scheme 2). Two methods were
applied to prepare 1a c (Scheme 2): HMDS activation at
elevated temperatures,[15a] in the presence of 1.3 equivalent of
acrylic derivative and subsequent quenching of the mixture of
proposed diastereomeric enolates (formed during the Michael
addition, Figure 4) with dry ethanol at À108C for 1 h,[25]
afforded the dipeptidic blocks 1 as a mixture of diastereomers
in a 3.5:1 ratio (Table 1, entries 1a (footnote a) and 1c).
Activation with TMSCl/DIPEA[36a] at low temperatures and
quenching of the mixture under the above described con-
ditions improved the diastereomeric ratio slightly (4.5:1, 1a,
footnote b). The ratio of the two diastereomers was easily
determined by 31P NMR spectroscopy. RP-HPLC could not
be used since no separation of the dipeptidic diastereomers
could be achieved at this stage. In the case of alanine
phosphinic analogue (1b) the ratio of diastereomers (3.5:1)
was lower than that obtained for the phenylalanine analogue,
presumably due to the smaller size of the methyl group. It
should be noted that use of bulky or chiral nonracemic
alcohols such as tert-butanol or (R)-a-amino phenyl glycinol
to quench the reaction mixture did not improve the obtained
ratios.
O
H
N
P
O
O
R1
O
P1' position
Figure 3. General structure of the phosphinic precursors and isoxazole-
containing peptidic inhibitors obtained by 1,3-dipolar cycloaddition
(above). Presumable interaction of these inhibitors with the active site of
zinc-metalloproteases through hydrogen bonding and/or coordination of
the isoxazole ring to the zinc cation (below).
prepared, containing a triple bond in this position (Scheme 1).
Construction of the isoxazole ring would be accomplished by
a [3 ‡ 2] cycloaddition of nitrile oxides to the alkyne moiety,
since the former are reported to add regioselectively to
terminal triple bonds giving exclusively 3,5-disubstituted
isoxazoles.[22]
Results and Discussion
Solution-phase synthesis: The retrosynthetic analysis of the
target pseudopeptide I is described in Scheme 1. The tripep-
tidic scaffolds II, containing a triple bond at P1' position, could
serve as good substrates for nitrile oxide III cycloadditions
(DCRS), allowing rapid access to the novel class of isoxazole
phosphinic peptides. This approach is consistent with our
initial goal and would allow the direct and straightforward
diversification of a tripeptidic unit at the final step of the
synthesis in one reaction step. Compounds II could be
synthesized by coupling of the pseudodipeptidic units IV with
amino acids V. Finally, phosphinic dipeptides IV could be
Saponification of the ethyl ester of 1 afforded the diacids
2a c. Alkaline removal of the ethyl ester of 1 was carefully
carried out, since large excess of hydroxide anions
(ꢀ10 equiv) and long reaction
times (ꢀ10 h) caused partial
R1
O
R2
R1
O
R2
O
O
racemization at the P1' position
(ꢀ3 4%). Accordingly, con-
trolled saponification (5 6 e-
quiv NaOH, TLC monitoring
X1
X2
X1
X2
N
H
P
N
N
H
P
N
H
R3
C
N O
H
+
OH
O
OH
O
O
III
N
I
II
R3
6
8 h) allowed minimization of
this undesired result. The major
diastereomer 2a' (R,S configu-
ration) of diacid mixture 2a
could be easily obtained by
simple recrystallization from
ethyl acetate. 31P NMR spectro-
scopy proved to be a very con-
R1
O
R1
O
O
O
O
X1
X1
X3
H2N
N
H
P
X2
N
H
PH
OH
X3
+
+
OH
R2
VII
IV
V
VI
Scheme 1. Retrosynthetic analysis of the isoxazole-containing tripeptidic inhibitors I.
Chem. Eur. J. 2003, 9, 2079 2094 www.chemeurj.org ¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2081

A. Yiotakis et al.
FULL PAPER
R1
we were able to succeed in a
more practical process and ach-
ieved our initial goal (isolation
of RSS diastereomer) by means
of simple crystallization from
appropriate solvent. This prop-
erty of tripeptides of the gen-
eral formula Cbz(R)XaaY-
[P(O)(OH)CH2]CH(propargyl)-
(S)TrpNH2 was successfully ex-
ploited to prepare peptides with
different side chains at P1 posi-
tion in very good yields and
R1
OTMS
P
O
Cbz
Cbz
i)
N
H
N
H
P H
(R)
(R)
R1
O
OTMS
OH
O
ii)
Cbz
N
H
P
O
(R)
O
OH
1a-c
O
1a: R1=CH2Ph
1b: R1=CH3
1c: R1=Ph
iii)
NH
R1
O
R1
O
O
O
iv)
(S)
Cbz
NH2
Cbz
N
N
H
P
N
(S)
P
OH
(R)
(R)
H
H
OH
O
excellent
optical
purities
OH
2a-c
3a-c
(ꢀ97% de) (Table 2).
Furthermore, the resolved
(R,S)-diastereomer 2a' turned
out to be a valuable tool for
many reasons. It could be easily
coupled with amino acids to
afford an array of propargyl-
containing tripeptidic blocks di-
versified at P2' position (e.g.
entry 3d, Table 2). To the best
of our knowledge, only very few
examples of diastereomeric
phosphinic dipeptides have
been successfully resolved at
this stage.[6] We utilized this
O
O
O
O
v)
vi)
(S)
(S)
Cbz
Cbz
O
2a
N
H
P
OH
N
H
P
N
H
(S)
(R)
(R)
OH
OH
O
2a'
3d
Scheme 2. Diastereoselective preparation of the tripeptidic units 3, suitable for 1,3-dipolar cycloaddition with
nitrile oxides. i) HMDS (5 equiv), 1108C, 1 h (activation); or TMSCl (4.5 equiv), DIPEA(4.5 equiv), 0 8C ! RT,
3 h (activation); ii) 1008C, 3 h (Michael addition), then dry EtOH, À108C ! RT; or 0 8C ! RT, 48 h (Michael
addition), then abs. EtOH, À108C ! RT; iii) 0.5m NaOHaq/EtOH (5 equiv), 08C ! RT, 6 8 h, then 1m HCl to
pH ꢀ 1; iv) HCl ¥ HTrpNH2 (1 equiv), EDC ¥ HCl (4 equiv), HOBt (1 equiv), DIPEA(3 equiv), CH 2Cl2, 1.5 2 h,
crystallization in AcOEt or CH2Cl2; v) recrystallization in AcOEt; vi) HCl ¥ HAlaOMe (1 equiv), EDC ¥ HCl
(4 equiv), HOBt (1 equiv), DIPEA(3 equiv), CH Cl2, 1.5 2 h.
2
advance in our studies regarding racemization at the P1'
position throughout our project, since we were not aware of
any other relevant studies. Notably when 2a' was coupled with
alanine methyl ester to afford tripeptide block 3d, no
racemization was observed under these conditions (EDC ¥
HCl, HOBt, short reaction time) according to HPLC analysis
(Scheme 2, Table 2).
OSiMe3
OEt
OEt
R1
R1
O
P
O
P
OSiMe3
Cbz
(R)
Cbz
(R)
N
H
N
H
OH
OH
(R, E)-enolate
(R, Z)-enolate
Figure 4. Proposed diastereomeric enolates formed during the Michael-
type addition of silyl phosphonites to acrylic derivatives.
Before we embarked on the DCRs to these precursors, it
was essential to unambiguously establish the absolute config-
uration of the newly formed stereogenic carbon (P1' position).
Aliterature search regarding RP-HPLC analysis of phos-
phinic pseudotripeptides, containing an (S)-amino acid (P2'
position) linked to P1' position of the phosphinic dipeptidic
unit by a classical amide bond and therefore existing as
mixture of two diastereomers (e.g. R, R/S, S), indicated that
the isomer with the shorter retention time is the one
Table 1. Methods, yields and ratio of diastereomers of the dipeptidic units
of type 1.
Entry
R1
Yield [%]
Ratio (R,S)/(R,R)[c]
1a[a]
1a[b]
1b
CH2Ph
CH2Ph
Me
96[a]
89[b]
92[b]
88[a]
3.5.1
4.5:1
3.5:1
3.5:1
1c
Ph
[a] HMDS-method. [b] TMSCl/DIPEA-method. [c] Based on 31P NMR;
for determination of the absolute configuration see Scheme 3.
Table 2. Tripeptides of type 3.
venient means for monitoring recemization during the ethyl
ester hydrolysis or the progress of attempted resolution of the
dipeptidic units 2. For diacids 2b,c such a resolution could not
be accomplished. Nevertheless, this result did not impact the
progress of our project, since tripeptide mixtures 3a c were
easily resolved by fractional crystallization.
The determination of the absolute configuration of the new
stereogenic center was accomplished after coupling of the
dipeptides 2 with (S)-tryptophan amide to afford the tripep-
tides 3a c (Table 2). Although the diastereomeric mixtures 3
could be easily separated into its components by RP-HPLC,
Entry
R1
R2
P1
Yield [%]
3a
3b
3c
3d
CH2Ph
Me
Ph
3-indolylmethyl[a,b]
3-indolylmethyl[a,c]
3-indolylmethyl[a,b]
Me
NH2
NH2
NH2
OMe
79
73
82
79
CH2Ph
[a] Combined yields of (R,S,S) and (R,R,S) diastereomers, starting from
the appropriate dipeptide as mixture of diastereomers. [b] Recrystalliza-
tion in AcOEt (ꢀ98% de). [c] Recrystallization in CH2Cl2 (97% de).
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Combinatorial Array Synthesis
2079 2094
possessing the (S) absolute con-
figuration at the P1' posi-
tion.[4e, 6, 25, 26] In our case, given
the (R) and (S) configuration of
the amino phosphinic acid and
tryptophan, respectively, we ob-
served two tripeptidic diaster-
eomers, with the firstly eluted in
RP-HPLC formed in much high-
er percentage than the other one.
Based on the above mentioned
literature results, we surmised
that the first fraction is the
(R,S,S) diastereomer, while the
second is the (R,R,S) one.
To unambiguously establish
this hypothesis, we sought to
obtain further evidence. To this
end, we applied the 1H NMR
method of Chen et al.[6b] for the
two diastereomers of phosphinic
tripeptide 3a, isolated by means
of simple crystallisation [besides
the insoluble isomer of this tri-
peptide mixture (assumed to be
the (R,S,S) diastereomer judging
from HPLC analysis), the solu-
ble isomer (assumed as the
(R,R,S)) was also recovered
from the filtrate, at least 95%
(HPLC analysis) and in high
optical purity (ꢀ93% de)].
According to Chen×s method,
in a dipeptidic unit containing
one aromatic residue, the rela-
tive absolute configuration of
each amino acid could be de-
duced from the chemical shifts of
the nonaromatic side chain pro-
tons: a shielding of these protons
is observed when the two amino
acids have opposite absolute
configuration (R,S or S,R) as
compared to those having iden-
tical configuration (R,R or S,S).
In our case, the existing trypto-
phan residue at P2' position of
the tripeptides rendered this
Scheme 3. Correlation between characteristic chemical shifts of propargyl and allyl side chain (P1' position)
protons and absolute configuration of these chains in phosphinic tripeptides precursors to DCRs. The 1H NMR
spectra were acquired using the crude hydrogenated products.
method
highly
relevant.
1H NMR analysis of the two diastereomers clearly showed,
in accordance with the published method, deshielding of the
propargylic proton in the ™(R,S,S)∫ isomer by ꢀ0.14 ppm, as
compared to the propargylic proton of the ™(R,R,S)∫ isomer
(2.74 vs 2.60 ppm respectively confirmed by HMQC experi-
ments, Scheme 3). This result confirmed the assumed absolute
configuration of the P1' position.
In the course of our program aiming at the development of
novel heterocyclic phosphinic peptides, it occurred to us that
our results could be ultimately confirmed by applying the
same method to the analogue of tripeptide 3a, bearing an
allylic instead of a propargylic side chain (4a, Scheme 3).
Isolation of such a tripeptide was achieved in the same fashion
as for tripeptide 3a. Crystallisation from ethyl acetate
afforded the desired (R,S,S) diastereomer (4a, Scheme 3), as
proven by 1H NMR analysis. Indeed, 1H NMR analysis of the
mixture of the two diastereomers of 4 (a mixture with a 3.5:1
unoptimised ratio by HPLC analysis) showed two multiples,
due to the single allylic proton at ꢀ5.2 and 5.5 ppm,
respectively, in a 3.5:1 ratio. Moreover, in the 1H NMR
Chem. Eur. J. 2003, 9, 2079 2094
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A. Yiotakis et al.
FULL PAPER
spectrum of the pure assumed (R,S,S) diastereomer (4a), only
the peak of the most deshielded proton (5.5 ppm) was
retained, leading to the conclusion that the isolated isomer
indeed possessed the (R,S,S) configuration.
hydroxyphosphinyl functionality; in addition the troublesome
removal of the by-product diphenylurea further rendered this
method problematic. Next, we turned our attention to the
phase transfer catalyzed (PTC) hypochloride method by
Lee[31] that consists in the use of (common) bleach (NaOClaq)
in CH2Cl2/cat. Et3N. Although this convenient one-pot
procedure worked very well for simple dipeptides (e.g. 1a,
data not shown), it failed in the case of the tryptophan-
containing tripeptidic blocks 3a c. Specifically, upon addi-
tion of bleach into the tripeptide solution, oxidative destruc-
tion of the indole ring was observed leading to a mixture of
unidentified products. Unexpectedly, in the case of tripeptide
3d, this method failed again for a different reason; HPLC
analysis of the isolated mixture indicated extensive removal of
the methyl ester of alanine residue, traces of starting material
and some product. It is likely that under the alkaline
conditions of this method the sensitive methyl ester was
saponified; this unexpected hydrolysis might be related to the
position of the ester inside the peptidic sequence (P2'
position), since ethyl ester removal, when present at P1'
position (e.g. 1a), was not observed at all under the same
conditions.
The Huisgen method was the third option chosen for
evaluation. While the two above mentioned methods are one-
step procedures, the Huisgen method can be performed as
either one- or two-step procedure depending on the nature of
the oxime used and the volition of the chemist.[32] This
versatile method consists of prior conversion of an oxime into
its hydroximinoyl chloride by action of N-chlorosuccinimide
(NCS), isolation of this chloride, and subsequent slow
generation of the corresponding nitrile oxide by means of
base-induced dehydrochlorination in the presence of a
dipolarophile (triple bond in this work).
With this pure diastereomer in hand, we were ready to
reconfirm our results for the propargyl-containing tripeptide
3a. We thought that hydrogenation of the triple bond of any
diastereomer of 3a, in the presence of Lindlar catalyst,[27]
could afford the corresponding tripeptides 4a or 4a'. This
proved to be the case, thus allowing a direct comparison and
evaluation of the two diastereomeric tripeptides. This con-
version is depicted in Scheme 3, where the (R,S,S) propargylic
diastereomer 3a afforded, after controlled hydrogenation, the
corresponding allylic diastereomer 4a (85% product, 10%
starting tripeptide, 5% fully hydrogenated tripeptide after 4 h
of hydrogenation, HPLC analysis), still suitable for NMR
analysis. The same motif was observed when the (R,R,S)
diastereomer was subjected to hydrogenation (product 4a'). It
1
should be noted that assessment of the H NMR spectrum is
much more convenient for the allylic derivatives, since their
protons are greatly deshielded as compared to propargylic
protons. Comparison of the spectra of these compounds
demonstrated unequivocally their configurational identity.
Having developed reliable methods for the preparation of a
wide range of optically pure propargylic phosphinic tripep-
tides, we were in position to study their reactivity toward 1,3-
dipolar cycloadditions (DCRs) with nitrile oxides. Although
such additions onto activated and nonactivated triple bonds
have been extensively studied and applied to incorporate the
isoxazole ring into numerous organic compounds ranging
from natural products to amino acids analogues and low
molecular weight non peptide bioactive compounds, they
have been only sparingly used in peptidic modulation.[28]
The chemistry of phosphinic pseudopeptides is subject to
many restrictions compared with classical peptides, mainly
due to the hydroxyphosphinyl moiety. Without precedent for
DCRs in the phoshopeptide field, the evaluation of different
isoxazole ring-constructing formation methods was essential
(Figure 5). The isolated, nonactivated nature of the triple
bond in our peptidic precursors was expected to render them
fairly good substrates for DCRs.[29]
Indeed, applying a one-pot Huisgen procedure we were
able to perform DCRs of a wide range of nitrile oxides onto
the phosphinic tripeptidic dipolarophiles 3a d. Chlorination
of oximes was performed using NCS in chloroform, pyridine
as catalyst, and gentle heating (method A, Scheme 4). The
chlorination time for benzaldoximes is dependent on the
nature of the ring substitution and for strongly deactivated
oximes longer times (ꢀ3 h) and heating are necessary to
achieve chlorination. The same method was successfully
At the outset, the Mukaiyama method[30] (primary nitro-
alkanes/phenyl isocyanate/Et3N), though extensively applied
in DCRs, was deemed incompatible with the unprotected
R1
O
R2
O
O
O
L
(S)
P1
i) or ii)
Cbz
(S)
NOH
P
+
(S)
(R)
N
H
P
N
OH
R3
H
N
OH
Et3N
OH
O
O
syn or anti
N
3a-d
5a-i
Cl
R
L=H,Cl
R3 (see Table 3)
R3
Huisgen method
3,5-disubstituted
isoxazole
(see Figure 2)
OH
N
R'
NaOCl
iii)
R
C N O
+
Mixture of by-products
H
R
O
O
Scheme 4. Methods for the preparation of the isoxazole-containing
phosphinic pseudopeptides of type 5. i) Method A(one-flask procedure):
L ˆ H, oxime (6 equiv) in CHCl3, pyridine(cat.), NCS (6 equiv), 458C, 3
4 h, then 3a d, Et3N (7 equiv, portionwise), 458C, 48 60 h. ii) Method B
(two-step procedure): L ˆ Cl, oxime (6 equiv) in DMF, NCS (6 equiv),
458C, 40 min, isolation of the hydroximinoyl chloride, then 3a, Et3N
(7 equiv, portionwise), RT, 48 h. iii) NaOClaq, CH2Cl2, Et3N (cat.), RT,
12 h.
N
N
PTC hypohalide method
R
R
PhNCO
Et3N
Mukaiyama method
furoxan dimer
RCH2NO2
Figure 5. Methods of nitrile oxide generation.
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Combinatorial Array Synthesis
2079 2094
applied for chlorination of naphthaldoxime or heterocyclic
oximes such as 2-furyl aldoxime. It is worth mentioning that
the NCS chlorination of one isomer (syn or anti, stereo-
chemistry undetermined) of the oximes was very rapid, while
several hours of reaction were necessary to complete the
chlorination of the other isomer. When the chlorination was
over, a one-flask procedure was followed adding the tripepti-
dic dipolarophile 3a d and Et3N. We found that we could,
conveniently, perform the DCRs at ꢀ458C, thus raising the
yield of the target molecules (5) by about 10% (Table 3).
For alkyl aldoximes thisx synthesis gave unsatisfactory
results, which led us to modify the procedure. The best course
of action proved to be a two-step Huisgen method.[33] Thus,
the alkyl aldoxime was chlorinated in DMF for a short time
(ꢀ40 min) and the alkyl hydroximinoyl chloride was isolated
by extraction. Once more, the in situ base-generated highly
unstable alkyl nitrile oxide species was trapped by the
dipolarophile at RT, leading to the smooth preparation of
the isoxazole-containing tripeptide in satisfactory yield
(Scheme 4, method B, Table 3, entry 5d, see also [b] in this
Table).
tion. Moreover, it is applicable and high yielding for
benzaldoxime or benzaldoximes with electron-donating sub-
stituents and naphthaldoxime (76 92%). For heterocyclic
aldoximes such as 2-furyl aldoxime, benzaldoximes with
electron-withdrawing substituents and alkyl aldoximes still
worked well, giving rise to the target molecules in moderate to
good yields (55 64%). In these cases, the starting tripeptide
could easily be isolated from the reaction mixture (simple
extraction) and resubjected to DCRs with the same nitrile
oxide, allowing thus the preparation of the desired tripeptide
in higher yield (e.g. Table 3, entry 5 f). According to ESMS
analysis of all final compounds (5a i) no ring-chlorinated
material was detected. Furthermore, it must be underscored
that the addition of all nitrile oxides to the terminal triple
bonds was completely regioselective, affording only the
desired 3,5-disubstituted isoxazoles[22] (NMR analysis); in
addition, the integrity of the stereogenic carbons of the
starting tripeptides was not affected under the alkaline
conditions applied as concluded by HPLC analysis. In our
project some model tripeptides (Table 3, entries 5a,e,i) were
prepared as mixture of epimers at P1' position, in order to
reevaluate the inhibitory potency of the individual diaster-
eomers after HPLC separation.
By inspection of Table 3, it becomes apparent that our
approach to the synthesis of this novel heterocyclic class of
phosphinic pseudopeptides is both versatile and promising.
Amongst its merits, we should include for sure the ability of
diversification of the tripeptidic blocks at the final stage,
without the need for any hydroxyphosphinyl moiety protec-
Solid-phase synthesis: Solid-phase synthesis has been the
major tool for combinatorial chemistry with apparent advan-
tages over solution-phase chemistry. The application of solid-
phase organic synthesis (SPOS) in the preparation of small
compound libraries and peptides is of great significance in the
discovery and development of new drug compounds.[34] Syn-
thesis of phosphinic peptides has been successfully accom-
plished on the solid phase as well, using either parallel or
combinatorial strategies. Such an approach has led to the
rapid preparation and identification of highly potent and
selective inhibitors of various zinc-metalloproteases.[3b, 4a,c, 7b,
Table 3. Isoxazole-containing tripeptides of type 5.
Entry R1
R2
R3
P1
Yield [%]
86[a]
c, 12, 15a, 35]
These synthetic routes rely mainly on the use of a
building block approach.[36] The past few years have also
witnessed explosive developments in the solid-phase synthesis
of heterocyclic derivatives.[37] The isoxazole ring undoubtedly
belongs to the class of compounds whose solid-phase con-
struction has been extensively studied.[34, 37, 38]
5a
5b
CH2Ph 3-indolylmethyl
CH2Ph 3-indolylmethyl
NH2
NH2
78
5c
CH2Ph 3-indolylmethyl
NH2
NH2
76
Following the same trend, we were interested in developing
conditions, which would allow construction of the final
isoxazole-containing phosphinic inhibitors on the solid sup-
port. Were such an approach successfully developed, it would
prove to be very convenient for the rapid construction of the
target molecules in quantities sufficient for biological evalua-
tion. Moreover, during a solid-phase synthesis of isoxazoles,
one can with advantage use a large excess of nitrile oxides to
drive the DCRs to completion, without being concerned
about the removal of this excess from the target compounds.
On the contrary, solution-phase synthesis of isoxazoles is
often problematic due to the high propensity of nitrile oxides
to undergo dimerization to furoxan N-oxide, which renders
both isolation and purification of the desired compound
difficult.[22] In addition, development of a solid-phase version
of our strategy for the preparation of this novel class of
phosphinic peptides would set the first example of post-
modification in this field.
5d
5e
CH2Ph 3-indolylmethyl
CH2Ph Me
55[b]
OMe 81[a]
5 f
5g
5h
5i
CH2Ph Me
OMe 58 (87)[c]
CH3
Ph
3-indolylmethyl
3-indolylmethyl
3-indolylmethyl
NH2
NH2
NH2
91
92
Ph
64[a]
[a] Prepared as mixture of two diastereomers at P1' position; yield refers to
the mixture of isomers. [b] Prepared by method B (see text and Exper-
imental Section). The moderate yield is, in part, due to the relatively high
solubility of the product in the organic solution used to precipitate and
purify. [c] In parenthesis: overall yield after a second 1,3-dipolar addition to
the isolated starting tripeptide.
Chem. Eur. J. 2003, 9, 2079 2094
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2085

A. Yiotakis et al.
FULL PAPER
To date, solid-phase synthesis of
phosphinic peptides has been real-
ized mainly through the building
block approach and in all cases, the
used blocks lack of any side-chain
functionality.[36] Another issue often
addressed concerns the necessity of
prior protection of the hydroxy-
phosphinyl moiety.[15a] Most syn-
thetic schemes rely on the use of
P-protected precursors; however,
coupling of phosphinic dipeptidic
blocks bearing an unprotected hy-
droxyphosphinyl moiety to solid-
phase support has very briefly re-
ported only once.[39] The use of
P-unprotected dipeptidic blocks of-
fers a few advantages such as fewer
synthetic steps and easier analysis of
the intermediates, but on the other
hand it involves by-product forma-
tion because of the hydroxyphos-
phinyl moiety activation under the
reaction conditions.[16] In our case,
due to the existence of a triple bond
i)
ii)
L-series
pins
Rink-NHFmoc
Rink-NH2
=
i)
Rink-NH-CO-TrpFmoc
Rink-NH-CO-Trp-NH2
6a
6b
O
O
iii)
v)
iv),
Cbz
Rink-NH-CO-Trp-NH
P
(R)
OH
N
H
7
O
O
O
P
O
P
Cbz
vi)
Cbz
Rink-NH-CO-Trp-NH
N
H
TrpNH2
N
H
(R)
(R)
OH
OH
O
N
O
9
8
N
R4
R4
Scheme 5. Solid-phase synthesis of the isoxazole-containing phosphinic tripeptide array of type 9: i) 20% pip/
DMF; ii) FmocTrpOH, DIC, HOBt, CH2Cl2/DMF; iii) 2a or 2a', EDC¥ HCl, HOBt, DIPEA, CH2Cl2, RT, 2 h;
iv) R CH NOH (oxime), NCS, Py (cat.), CHCl3, 458C; v) Et3N, 45 8C; vi) TFA/H2O/TIS (95:2.5:2.5).
4
ˆ
not all protection schemes present orthogonality (e.g. hydro-
genation of Cbz group would lead to simultaneous reduction
of the alkyne moiety). Moreover, introduction of the ada-
mantyl group into the phosphinic moiety[15a] proved a little
troublesome, in addition to the potential steric hindrance that
would exert to the subsequent DCRs. Therefore, we decided
to develop a method that would allow the use of a
P-unprotected dipeptidic block such as 2a (Scheme 1).
Another key-feature of our solid-phase synthetic effort is
the use–for first time in the phosphinic peptide field–of
Mimotopes SynPhase PS Lanterns (L-series ™pins∫).[40] Use of
multipin technology is emerging as a viable alternative to
conventional resin solid-phase synthesis and is gaining in
significance owing to its many significant fundamental
advantages over resins.[28, 41] Given our desire to obtain
C-terminal amides, we chose pins functionalized with a Rink
amide linker and set out to develop and evaluate strategies for
the preparation of a few-member combinatorial array of
isoxazole-containing phosphinic tripeptides of the general
type 9 (Scheme 5).
Our synthetic efforts (Scheme 5) commenced with coupling
of Fmoc-protected tryptophan with pin-supported Rink
amide linker under standard conditions to afford the inter-
mediate 6a. Piperidine-mediated removal of Fmoc protection
delivered 6b. At this point we had to establish a method for
coupling of the P-unprotected dipeptidic block 2a with amine
6b. The previously described method[39] for a similar coupling
was not thoroughly documented and described the simple
building block [Z(R)PheY[(P(O)(OH)CH2]GlyOH], and
therefore, if employed, would require optimization. Although
our solution coupling method has never been applied on the
solid phase, we were very much interested in testing and
evaluating its efficiency under solid-phase conditions. Special
care had to be taken because the use of pins imply a standard,
low loading (14 mmol per pin) and conditions applied in
solution or on resin experiments needed careful evaluation to
avoid both large excesses of reagents and overly dilute
reaction solutions. In addition, the existence of a free
hydroxyphosphinyl moiety called for further limitations.
Preliminary experiments using EDC/HOBt-mediated cou-
pling started by using the block 2a (not resolved at P1'
position). Use of one equivalent of dipeptidic block in several
solvent systems (e.g. DMF, DMF/CH2Cl2, CH2Cl2/THF)
proved completely unsuccessful, delivering the intermediate
7 in unacceptable yields (ꢀ50%, RP-HPLC analysis). The
best result was achieved using CH2Cl2 as solvent, probably
due to high swelling of pins.[41b] Therefore, all subsequent
experiments were carried out in this convenient solvent.
Extensive experimentation revealed that the best course of
action was double coupling using: 1.5 equivalent (per pin) of
2a, four equivalents of EDC ¥ HCl, 1.5 equivalent of HOBt,
2.5 equivalents of DIPEAin CH 2Cl2 (0.25 mL per pin) for 3 h.
This protocol allowed the smooth synthesis of 7 quantitatively
and in excellent purity. Parallel studies showed that the DIC
coupling reagent could perform such couplings but it gave
lower yields (ca. 20%) than the EDC/HOBt system. In
addition, when the coupling time was extended to 24 h partial
racemization occurred.
To examine this racemization problem during the EDC/
HOBt-mediated coupling, we used the optically pure dipep-
tidic block 2a'. Unfortunately, coupling of 2a' for 24 h gave
rise to an unexpectedly high racemization percentage
(ꢀ17%). We were not aware of any studies (concerning P1'
position) that could provide us with relevant data. Thereby,
reevaluation of our established protocol had to be considered
in order to minimize racemization in the case that optically
pure blocks were used. Fortunately, shorter reaction times
proved a very good remedy for this predicament. Thus, the
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Combinatorial Array Synthesis
2079 2094
Table 4. Characteristics of the members of the isoxazole-phosphinic peptide combinatorial array 9a k, prepared
by DCRs on pins.
standard protocol was still ap-
plicable, but two couplings for
exactly 2 h were necessary to
obtain the tripeptidic block 7 in
high yield and high optical
purity. Given the fact that the
starting dipeptidic block had an
optical purity ꢀ98% (see be-
low), the estimated racemiza-
tion by application of this modi-
fied protocol was about 1%.
This result was considered ac-
ceptable given the oligomeric
nature of phosphinic peptides
(only one phosphinic block in-
corporated in the peptidic se-
quence). PyBOP-mediated[39]
coupling of 2a' did not offer
any additional advantage.
The mechanism of coupling
of phosphinic dipeptides with
amino acids has not been stud-
ied and reported, but it may be
assumed that it involves the
prior formation of a relatively
hindered, and therefore more
susceptible to racemization
than a classical HOBt C-active
ester, mixed cyclic anhydride
intermediate, which is later on
attacked by the amino compo-
nent.[9a] The fact that no race-
mization at all was observed
under solution- versus solid-
phase condition couplings (see
above) may be related to some
special features of the micro-
Entry
R4
Yield[a] [%]
Purity[b] [%]
tR value[c]
[min][d]
MW
ES-MS
[M À H]À
9a
9b
82
69
87
89
27.4/28.8
28.9
747.2
783.2
746.3
782.3
9c
66
70
30.7/31.9
825.3
824.2
9d
9e
89
81
30.5/31.9
21.4/22.1
809.3
776.3
808.3
775.3
93 (54)[e]
71 (95)[e]
9 f
9g
9h
78
89
26.1/27.1
25.5/26.1
32.5/34.1
793.3
758.3
843.3
792.3
757.2
842.3
55[f]
81
61[f]
82
9i
9j
73
77
80
83
33.6/35.3
26.0
843.3
733.3
842.2
732.2
9k
65
81
26.9
811.2
810.1
[a] Based on the initial loading of pins (14 mmol per pin). [b] Concluded by combining area of product HPLC
peaks (254 and/or 280 nm detection). [c] Each HPLC analysis was carried out with the gradient described at the
experimental section, general procedures. [d] The double time value corresponds to the two diastereomers of the
product; in the case of optically pure inhibitor only one diastereomer was detected. [e] In parentheses yield and
purity after post-cleavage solution extraction. [f] Low yield and purity due to uncompleted DCR; the starting
tripeptide was also recovered in the TFA-cleaved mixture.
environment of the resin (e.g. more difficult attack of the pin-
attached amine onto the presumed cyclic mixed anhydride,
extended reaction time for formation of such an anhydride
etc.). The hypothesis of the cyclic anhydride versus classical
C-active ester formation is supported by the fact that
performance of the same coupling (EDC/HOBt protocol) in
the presence of copper(ii) chloride[42] did not cut down on
recemization as it has been reported, but actually decreased
the yield and increased the extent of racemization (ꢀ7%).
This was presumably the result of difficulty/delay in the
formation of the mixed anhydride due to potential coordina-
tion of copper to the phosphinyl moiety.[43]
illustrated in Figure 6. From this Figure it is obvious that the
best set of conditions for conducting DCRs is the use of a
0.56m chloroform solution (namely 10 equivalents per pin) of
in situ generated nitrile oxide and gentle heating (458C) for
24 h. Prolonged reaction times (e.g. 48 h) did not improve the
yield, probably due to dimerization of nitrile oxides after a
certain point; therefore all subsequent experiments were
conducted for 24 h.
Having assembled the tripeptidic unit 7 on solid support as
both optically pure and mixture of epimers at P1' position, we
focused our efforts on studying the incorporation of the
isoxazole core into these precursors. To this end, a one-pot
Huisgen method (see above) was applied. Optimization of
DCRs of nitrile oxides onto 7 was easily and rapidly made by
using multipin technology. The model used in these studies
was compound 9a (see below, R4 ˆ p-methyl phenyl, Table 4).
Several parameters were studied such as concentration of
nitrile oxide, temperature, reaction time. Our results are
Figure 6. Optimization of nitrile oxide DCRs onto the pin-supported
dipolarophilic tripeptidic unit 7. Concentration at this histogram (left) is
that of nitrile oxide generated in situ from the corresponding hydroxyimi-
noyl chloride. % Conversion was concluded by combining area of product
HPLC peaks.
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A. Yiotakis et al.
FULL PAPER
Table 5. Ki Values of some isoxazole-containing phosphinic peptides and RXP03 toward MMP-13 and MMP-14.
Application of this optimized
set of conditions allowed the
facile preparation of the de-
sired combinatorial array of
type 9. HPLC monitoring of
the progress of the DCRs in-
dicated that three to four repe-
titions of the addition were
necessary in order to achieve a
high enough percentage of con-
version and purity of the target
molecules. This course of action
afforded the peptides 9 in very
good yields and purities after
TFA-effected cleavage from
the solid support, as shown in
Table 4. The representative an-
alytical HPLC chromatogram
of the crude tripeptide 9b (Fig-
ure 7), clearly indicates the high
purity of the product. It is worth
mentioning that when optically
Compound
R
Diastereomer
Ki [nm] MMP-13
Ki [nm] MMP-14
RXP03
R,S,S
R,R,S
16
32
90
386
5a
5b
5c
R,S,S
R,R,S
1.6
160
2.5
330
R,S,S
R,S,S
14
28
20
5100
high potency displayed by compound 5a-(R,S,S) demon-
strates that the stereochemistry and the electronic properties
of the isoxazole ring are very well suited to interact with S1'
pocket of MMPs. This compound is one of the most potent
phosphinic inhibitors of MMP-14, well compared with re-
ported hydroxamic inhibitors such as Marimastat (Ki ˆ 1.8 nm
toward MMP-14).[12] Moreover, the fact that the compound
5a-R,S,S is at least 100-fold more potent than 5a-R,R,S,
reveals that the stereochemistry of the P1' side chain is critical
for potency in this series of isoxazole compounds. This critical
role of the P1' stereochemistry was no previously observed
with compound such as RXP03. This difference is probably
related to the presence of the isoxazole ring that involves
higher conformational restriction than aryl alkyl side chains.
In this respect, as shown by compound 5c, further substitution
of the isoxazole-attached ring may represent a strategy to
control the selectivity of the inhibitors. Indeed, compound 5c
is 180-fold more potent toward MMP-13 than MMP-14. This
issue is particularly important, as there is a high demand for
pure tripeptides were used as precursors to DCRs, no
racemization was observed. In the case of strongly deactivated
benzaldoximes such as 9g (p-CN phenyl, Table 4), the
addition was not driven to completion even after four
repetitions. Nevertheless, the desired peptide could be
isolated by post-cleavage (solution) extraction or semiprepar-
ative HPLC. All peptides of type 9 were identified by direct
comparison with solution phase produced peptides (e.g. 9b,j)
as well as by electrospray mass spectrometry (Table 4).
Biological Evaluation
At preliminary stage, some of the developed isoxazole-
containing phosphinic peptides were evaluated for their in
vitro activities to inhibit two MMPs: collagenase-3 (MMP-13),
which is over-expressed in breast carcinomas[44] and mem-
brane type-1 matrix metalloprotease (MT1-MMP, MMP-14),
a primary target to block pro-MMP-2 activation.[45] Our
results are summarized in Table 5.
47]
compounds able to block only a subset of MMPs.[45
Substitution of this kind, using the approach reported in this
study, will give access to various compounds with improved
potency and selectivity toward the MMPs. Full evaluation of
all prepared compounds is in progress and will be reported
soon.
Comparison of the Ki values of Table 5 clearly indicates that
isoxazole-containing phosphinic peptides are more potent
MMP inhibitors than alkyl-aryl analogues, such as RXP03.
For example, compound 5a-(R,S,S) is 36-fold more potent
toward MMP-14 than RXP03-(R,S,S); this indicates the
advantages of this novel class of phosphinic peptides. The
Conclusion
The efficient synthesis and res-
olution of phosphinic peptides
containing a propargylic side
chain at P1' position was descri-
bed. Their absolute configura-
tion was established using
NMR studies. These pseudo-
peptidic blocks were studied
Figure 7. Analytical RP-HPLC chromatogram of the crude phosphinic peptide 9b (one diastereomer).
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Combinatorial Array Synthesis
2079 2094
(Atheris Laboratories, Geneva, Switzerland). RP-HPLC analyses were
performed on a Hewlett Packard 1100 model (C18-Cromasil-RP, 5 mm, UV/
Vis detector, flow: 0.5 mLminÀ1, 254 and/or 280 nm detection), with the
gradient: 0 min: 0% B buffer, 10 min: 35% B buffer, 35 min: 100% B
for their reactivity toward 1,3-dipolar cycloaddition reactions
(DCRs) with nitrile oxides. To this end, different methods of
cycloadditions were evaluated, leading to the successful
development of a novel class of phosphinic peptides, incor-
porating an isoxazole ring at P1' position, in high yields and
optical purity. Extension of this strategy on solid-phase
support was achieved using multipin technology. Develop-
ment of a new racemization-free protocol for coupling of
P-unprotected dipeptidic blocks with solid-phase-embodied
amino acids and subsequent optimization of DCRs onto the
produced peptides allowed the preparation of a few-member
combinatorial array of isoxazole-containing phosphinic pep-
tides. The crude pseudopeptides obtained from the solid-
phase synthesis were of high purity according to RP-HPLC
analysis. Preliminary inhibition assays of these new hetero-
cyclic pseudopeptides demonstrated that they are very potent
inhibitors of MMPs.
buffer, 40 min: 35% B buffer (Abuffer: 90% H O with 0.1% TFA, 10%
2
CH3CN; B buffer: 10% H2O with 0.09% TFA, 90% CH3CN). Enzyme
assays and Ki values determination were performed as previously
described.[4b]
General procedures for Michael addition reaction
HMDS method: Amixture of Cbz N-protected a-aminophosphinic acid
(1 mmol) and HMDS (5 mmol) was heated at 1108C for 1 h under Ar, then
ethyl a-propargyl acrylate (1.3 mmol) was added dropwise. The resulting
mixture was stirred at 100 1058C for additional 3 h and slowly cooled to
RT. Then, the mixture was further cooled to À108C and dry ethanol was
added portionwise, still under Ar. The cooled mixture was stirred at this
temperature for 1 h and was left to reach RT. The solvent was removed
under vacuum and the residue was purified by column chromatography
(chloroform/methanol/acetic acid 7:0.3:0.3).
TMSCl/DIPEA method: DIPEA(4.5 mmol) and TMSCl (4.5 mmol) were
added under Ar to an ice-cold suspension of Cbz N-protected a-amino-
phosphinic acid (1 mmol) in dry CH2Cl2 (7 mL per mmol). The mixture was
stirred for 2 h at RT. Then, the solution was cooled to 08C, and ethyl a-
propargyl acrylate (1.3 mmol) was added dropwise. The mixture was stirred
at RT for 36 h under Ar. Then, it was cooled to À108C and dry ethanol was
added portionwise, still under Ar. The cooled mixture was stirred at this
temperature for 1 h and was left to reach RT. The solvents were removed
under vacuum and the residue was purified by column chromatography
(chloroform/methanol/acetic acid 7:0.3:0.3).
Current work in our laboratory is aiming at the develop-
ment of libraries of this class of phosphinic peptides, widely
diversified around the heterocyclic core and the P2' position.
Full report of inhibitory properties of these compounds
toward MMPs and other Zn-metalloproteases will be report-
ed in a forthcoming paper.
(1R)-1-{[(Benzyloxy)carbonyl]amino}-2-phenylethyl[2-(ethoxycarbonyl)-
pent-4-ynyl]phosphinic acid (1a): M.p. 113 1158C; Rf ˆ 0.68 (chloroform/
methanol/acetic acid 7:0.5:0.5). 1H NMR (200 MHz, [D6]DMSO/1%
CF3COOD): d ˆ 1.26 (t, 3J(HH) ˆ 6.8 Hz, 3H; CH2CH3), 1.78 2.18 (m,
Experimental Section
ꢁ
2H; PCH2), 2.38 3.18 (m, 6H; PCH2CH, CHHPh, CHHPh, CH2C CH,
Abbreviations: ACE-I: angiotensin converting enzyme I, AcOEt: ethyl
acetate, DCR(s): 1,3-dipolar cycloaddition reaction(s), DIC: N,N'-diiso-
propylcarbodiimide, DIPEA: N,N-diisopropylethylamine, DMF: N,N-di-
methyl formamide, EDC ¥ HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbo-
diimide hydrochloride, Et3N: triethylamine, HMDS: 1,1,1,3,3,3-hexame-
thyldisilazane, HOBt: N-hydroxy benzotriazole, MMPs: matrix
metalloproteases, NCS: N-chloro succinimide, Py: pyridine, PyBOP:
C CH), 3.81 4.19 (m and q, 3J(HH) ˆ 6.8 Hz, 3H; CH2CH3, PCH), 4.97 (s,
ꢁ
2H; PhCH2O), 7.06 7.38 (m, 10H; Ar), 7.72 (d, 3J(HH) ˆ 9.7 Hz, 1H;
NH); 13C NMR (50 MHz, [D6]DMSO/1% CF3COOD): d ˆ 14.1, 21.8, 26.9
(d, J(PC) ˆ 88.4 Hz), 32.6, 38.0, 52.2 (d, J(PC) ˆ 104.3 Hz), 60.5, 65.2, 73.2
(d, J(PC) ˆ 15.5 Hz), 80.9, 113.4, 126.2, 127.2, 128.2, 129.2, 137.2, 138.4 (d,
J(PC) ˆ 14 Hz), 156.1 (d, J(PC) ˆ 3.8 Hz), 172.9 (d, J(PC) ˆ 10.8 Hz); 31P
NMR (81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 45.0, 45.3, two diaster-
eomers: (R,S)/(R,R) ˆ 3.5/1 (1a, see Table 1), 4.5:1 (1a, see Table 1);
elemental analysis calcd (%) for C24H28NO6P: C 63.01, H 6.17, N 3.06;
found: C 62.71, H 5.92, N 2.81; ES-MS: m/z: calcd for C24H27NO6P: 456.2;
found: 456.3 [M À H]À.
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophos-
phate, RP-HPLC: reversed phase high performance liquid chromatogra-
phy, TFA: trifluoroacetic acid, THF: tetrahydrofuran, TIS: triisopropylsi-
lane, TMSCl: trimethylsilyl cloride, Y: symbol for peptide surrogates.
General procedures: Most anhydrous solvents were obtained by storing
analytical quality solvents over 4 ä activated molecular sieves. DMF and
THF were fractionally distilled (over P2O5 and CaH2 respectively) and
stored over 4 ä activated molecular sieves. Synthetic starting materials
were purchased from Aldrich, Fluka, NovaBiochem or Bachem and were
used without further purification. SynPhase PS Lanterns (L-series pins)
and Multipin stems were purchased from Mimotopes, Paris, France. All
aminophosphinic acids[24] and acrylic derivatives[23] were prepared accord-
ing to previously described methods. Oximes used in this work were
prepared from the corresponding aldehydes under standard procedur-
es.[32b, 48] Preparation and full characterization of compound 4a will be
reported elsewhere. Chemical reactions, washings and cleavages involving
pins were performed in chemically resistant glass tubes. 1H, 13C and 31P
NMR spectra were recorded on Varian spectrometers (200 MHz Mercury
or 300 MHz Gemini models). Assignment of the 1H NMR signals were
achieved using COSYand, in some cases only, HMQC experiments. 13C and
31P NMR spectra are fully proton decoupled. All spectra are referenced (d
scale in ppm) using the residual solvent as an internal standard (e.g. for
(1R)-1-{[(Benzyloxy)carbonyl]amino}ethyl[2-(ethoxycarbonyl)pent-4-
ynyl]phosphinic acid (1b): M.p. 95 978C; Rf ˆ 0.38 (chloroform/methanol/
acetic acid 7:0.5:0.5). 1H NMR (200 MHz, [D6]DMSO/1% CF3COOD):
d ˆ 1.22 (dd, 3J(HH) ˆ 7.1 Hz, 3J(PH) ˆ 15.8 Hz, 3H; CHCH3), 1.27 (t,
3J(HH) ˆ 6.8 Hz, 3H; CH2CH3), 1.68 1.93 (m, 1H; PCHH), 1.95 2.20 (m,
ꢁ
1H; PCHH), 2.47 2.62 (m, 2H; CH2C CH), 2.72 2.96 (m, 2H; PCH2CH,
C CH), 3.62 3.95 (m, 1H; PCH), 3.99 4.18 (q, 3J(HH) ˆ 6.8 Hz, 2H;
ꢁ
CH2CH3), 5.02 (s, 2H; PhCH2O), 7.23 7.39 (m, 5H; Ar), 7.61 (d, 3J(HH) ˆ
9.6 Hz, 1H; NH); 13C NMR (50 MHz, [D6]DMSO/1% CF3COOD): d ˆ
13.8, 14.1, 21.4, 26.2 (d, J(PC) ˆ 87.9 Hz), 37.7, 45.9 (d, J(PC) ˆ 105.4 Hz),
60.3, 65.6, 72.7, 81.1, 112.3, 118.0, 127.7, 127.8, 128.4, 137.2, 156.3 (d, J(PC) ˆ
3.9 Hz), 172.7 (d, J(PC) ˆ 10.5 Hz); 31P NMR (81 MHz, [D6]DMSO/1%
CF3COOD): d ˆ 45.8, 46.0, two diastereomers (R,S)/(R,R) ˆ 3.5/1; ele-
mental analysis calcd (%) for C18H24NO6P: C 56.69, H 6.34, N 3.67; found:
C 56.51, H 6.28, N 3.52.
(R)-{[(benzyloxy)carbonyl]amino}phenylmethyl[2-(ethoxycarbonyl)pent-
4-ynyl] phosphinic acid (1c): M.p. 98 1008C; Rf ˆ 0.55 (chloroform/
methanol/acetic acid 7:0.5:0.5); 1H NMR (200 MHz, [D6]DMSO/1%
CF3COOD): d ˆ 1.21 (t, 3J(HH) ˆ 6.9 Hz, 3H; CH2CH3), 1.65 1.94 (m,
1
[D6]DMSO; d ˆ 2.50, H and d ˆ 39.50, 13C). 31P NMR chemical shifts are
reported in ppm downfield from 85% H3PO4 (external standard). Column
chromatography was performed on silica gel (E. Merck, 70 230 mesh).
TLC analyses were performed on silica gel plates (E. Merck silica gel 60
F254) and components were visualized by the following methods: UV light
absorbance, and/or charring after spraying with a solution of NH4HSO4,
and/or ninhydrin spraying and heating. Melting points (measured on a
Electrothermal apparatus) are uncorrected. Electron spray mass spec-
trometry (ESMS) was performed on a Micromass Platform II instrument
ꢁ
1H; PCHH), 1.96 2.22 (m, 1H; PCHH), 2.42 2.65 (m, 2H; CH2C CH,
ꢁ
[D6]DMSO overlapping), 2.70 2.95 (m, 2H; PCH2CH, C CH), 3.99 4.20
(q, 3J(HH) ˆ 6.8 Hz, 2H; CH2CH3), 4.83 5.12 (m, 3H; PhCH2O, PCH),
7.17 7.58 (m, 10H; Ar), 7.82 (d, 3J(HH) ˆ 9.6 Hz, 1H; NH); 13C NMR
(50 MHz, [D6]DMSO/1% CF3COOD): d ˆ 14.1, 21.7 (d, J(PC) ˆ 7.6 Hz),
26.9 (d, J(PC) ˆ 92.1 Hz), 54.8 (d, J(PC) ˆ 100.2 Hz), 60.4, 65.5, 73.1, 80.8,
113.3, 126.2, 127.1, 128.5, 129.2, 137.3, 138.4 (d, J(PC) ˆ 14 Hz), 155.3 (d,
Chem. Eur. J. 2003, 9, 2079 2094
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2089

A. Yiotakis et al.
FULL PAPER
J(PC) ˆ 3.8 Hz), 172.7 (d, J(PC) ˆ 11.3 Hz); 31P NMR (81 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 42.1, 42.4, two diastereomers (R,S)/
(R,R) ˆ 3.5/1; elemental analysis calcd (%) for C23H26NO6P: C 62.30, H
5.91, N 3.16; found: C 62.22, H 6.18, N 3.33.
(2S)-2-({[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}carbo-
nyl)pent-4-ynyl
((1R)-1-{[(benzyloxy)carbonyl]amino}-2-phenylethyl)-
phosphinic acid (3a): M.p. 237 2388C; Rf ˆ 0.41 (chloroform/methanol/
acetic acid 7:2:1); 1H NMR (200 MHz, [D6]DMSO/1% CF3COOD): d ˆ
ꢁ
1.78 2.13 (m, 2H; PCH2), 2.23 2.58 (m, 2H; CH2C CH,), 2.60 2.97 (m,
General procedure for removal of ethyl ester: Dipeptides 1a c (1 mmol)
were dissolved in ethanol (9 mL per mmol), and the solution was cooled to
08C. 1m NaOH (5 6 mmol) was added portionwise and the mixture was
stirred at RT for 6 8 h. After acidification with 2m HCl, ethanol was
evaporated, diluted with water, and extracted with AcOEt. The organic
phase was washed with water and brine, dried over Na2SO4 and evaporated
ꢁ
3H; PCH2CH, CHHPh, C CH), 2.99 3.23 (m, 3H; CHHPh, CH2indolyl),
3.84 4.09 (m, 1H; PCH), 4.36 4.49 (m, 1H; CHCONH2), 4.96 (s, 2H;
PhCH2O), 6.83 7.38 and 7.40 7.59 (m, 18H; Ar, indolyl, NH2), 7.69 (d,
3J(HH) ˆ 9.7 Hz, 1H; NHCHP), 8.11 (d, 3J(HH) ˆ 9.7 Hz, 1H;
NHCHCONH2); 13C NMR (50 MHz, [D6]DMSO/1% CF3COOD): d ˆ
22.2, 27.8 (d, J(PC) ˆ 86.7 Hz), 32.7, 38.2, 52.4 (d, J(PC) ˆ 108.4 Hz), 53.5,
65.2, 72.7 (d, J(PC) ˆ 19.7 Hz), 81.9, 110.5, 111.2, 112.3, 118.2, 120.7, 123.6
(d, J(PC) ˆ 15.2 Hz), 126.3, 126.9, 127.2, 127.4, 128.2, 128.9, 136.1, 138.3,
138.4 (d, J(PC) ˆ 13.6 Hz), 156.1 (d, J(PC) ˆ 3.8 Hz), 172.2 (d, J(PC) ˆ
8.3 Hz), 173.4; 31P NMR (81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 45.9
(46.6 for (R,R,S) diastereomer). HPLC: tR ˆ 22.3 min (23.3 min for (R,R,S)
diastereomer); elemental analysis calcd (%) for C33H35N4O6P: C 64.49, H
5.74, N 9.12; found: C 64.31, H 5.65, N 9.01; ES-MS: m/z: calcd for
C33H34N4O6P: 613.2; found: 613.2 [M À H]À.
under vacuum. Precipitation from
a mixture of diethyl ether/light
petroleum (1:4) afforded the desired diacids as white solids.
(2S)-2-{[((1R)-1-{[(benzyloxy)carbonyl]amino}-2-phenylethyl)(hydroxy)-
phosphoryl] methyl}pent-4-ynoic acid (2a'): Recrystallization of the
precipitated diacid from AcOEt (30 mL per mmol) afforded the pure
diastereomer, yield: 96% (for saponification), (70% based on the pure
diastereomer); M.p. 176 1788C; Rf ˆ 0.21 (chloroform/methanol/acetic
acid 7:0.5:0.5); 1H NMR (200 MHz, [D6]DMSO/1% CF3COOD): d ˆ
1.76 2.18 (m, 2H; PCH2), 2.38 2.97 (m, 5H; PCH2CH, CHHPh,
(2S)-2-({[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}carbo-
ꢁ
ꢁ
CH2C CH, C CH), 2.99 3.17 (m, 1H; CHHPh), 3.81 4.05 (m, 1H;
PCH), 4.96 (s, 2H; PhCH2O), 7.05 7.41 (m, 10H; Ar), 7.76 (d, 3J(HH) ˆ
9.7 Hz, 1H; NH); 13C NMR (50 MHz, [D6]DMSO/1% CF3COOD): d ˆ
21.6, 26.8 (d, J(PC) ˆ 88.8 Hz), 32.6, 38.2, 52.4 (d, J(PC) ˆ 104.6 Hz), 65.2,
73.4, 80.7, 113.4, 126.3, 127.2, 128.2, 129.3, 137.2, 138.3 (d, J(PC) ˆ 13.8 Hz),
157.2 (d, J(PC) ˆ 3.8 Hz), 174.1 (d, J(PC) ˆ 10.6 Hz); 31P NMR (81 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 45.7 (45.3 for 2a); elemental analysis
calcd (%) for C22H24NO6P: C 61.54, H 5.63, N 3.26; found: C 61.67, H 5.48,
N 3.43.
nyl)pent-4-ynyl
((1R)-1-{[(benzyloxy)carbonyl]amino}ethyl)phosphinic
acid (3b): M.p. 181 1838C; Rf ˆ 0.25 (chloroform/methanol/acetic acid
7:2:1); 1H NMR (200 MHz, [D6]DMSO/1% CF3COOD): d ˆ 1.20 (dd,
3J(HH) ˆ 7.3 Hz, 3J(PH) ˆ 14.3 Hz, 3H; CHCH3), 1.68 2.05 (m, 2H;
ꢁ
PCH2), 2.22 2.44 (m, 2H; CH2C CH), 2.63 2.93 (m, 2H; PCH2CH,
ꢁ
C CH), 2.96 3.28 (m, 2H; CH2indolyl), 3.63 3.87 (m, 1H; PCH), 4.28
4.43 (m, 1H; CHCONH2), 5.04 (s, 2H; PhCH2O), 6.90 7.59 (m, 14H; Ar,
indolyl, NHCHP, NH2), 8.05 (d, 3J(HH) ˆ 7.6 Hz, 1H; NHCHCONH2);
13C NMR (70 MHz, [D6]DMSO/1% CF3COOD): d ˆ 14.0, 22.3 (d, J(PC) ˆ
9.2 Hz), 27.1 (d, J(PC) ˆ 89.4 Hz), 27.2, 46.0 (d, J(PC) ˆ 103.2 Hz), 53.6,
65.6, 72.6, 81.9, 110.4, 111.2, 118.3 (d, J(PC) ˆ 15.9 Hz), 120.8, 123.5, 127.4,
127.7, 127.8, 128.3, 136.1, 137.0, 155.8 (d, J(PC) ˆ 4.8 Hz), 172.1 (d, J(PC) ˆ
8.1 Hz), 173.3; 31P NMR (81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 46.6.
HPLC: tR ˆ 18.5 min (19.7 min for (R,R,S) diastereomer); elemental
analysis calcd (%) for C27H31N4O6P: C 60.22, H 5.80, N 10.40; found: C
60.33, H 5.89, N 10.21; ES-MS: m/z: calcd for C27H30N4O6P: 537.2; found:
537.4 [M À H]À.
2-{[((1R)-1-{[(benzyloxy)carbonyl]amino}ethyl)(hydroxy)phosphoryl]me-
thyl}pent-4-ynoic acid (2b): Yield: 92%; M.p. 149 1518C; Rf ˆ 0.32
(chloroform/methanol/acetic acid 7:2:1); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.22 (dd, 3J(HH) ˆ 7.1 Hz, 3J(PH) ˆ
14.1 Hz, 3H; CHCH3), 1.70 1.92 (m, 1H; PCHH), 1.96 2.20 (m, 1H;
ꢁ
PCHH), 2.47 2.62 (m, 2H; CH2C CH), 2.75 2.96 (m, 2H; PCH2CH,
ꢁ
C CH), 3.65 3.96 (m, 1H; PCH), 5.03 (s, 2H; PhCH2O), 7.21 7.40 (m,
5H; Ar), 7.61 (d, 3J(HH) ˆ 9.7 Hz, 1H; NH); 13C NMR (50 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 13.8, 21.5, 26.2 (d, J(PC) ˆ 87.2 Hz),
37.7, 45.7 (d, J(PC) ˆ 105.2 Hz), 65.5, 72.8, 81.3, 112.3, 118.1, 127.7, 127.8,
128.4, 137.0, 158.4 (d, J(PC) ˆ 3.8 Hz), 174.2 (d, J(PC) ˆ 10.3 Hz); 31P NMR
(81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 46.3, 46.9, two diastereomers
(R,S)/(R,R) ˆ 3.5/1; elemental analysis calcd (%) for C16H20NO6P: C 54.39,
H 5.71, N 3.96; found: C 54.51, H 5.88, N 3.83.
(2S)-2-({[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}carbo-
nyl)pent-4-ynyl [(R)-{[(benzyloxy)carbonyl]amino}(phenyl)methyl]phos-
phinic acid (3c): M.p. 197 1998C; Rf ˆ 0.39 (chloroform/methanol/acetic
acid 7:2:1); 1H NMR (200 MHz, [D6]DMSO/1% CF3COOD): d ˆ 1.63
ꢁ
2.13 (m, 2H; PCH2), 2.18 2.63 (m, 2H; CH2C CH, [D6]DMSO over-
ꢁ
lapping), 2.65 2.91 (m, 2H; PCH2CH, C CH), 2.94 3.32 (m, 2H;
2-{[[(R)-{[(benzyloxy)carbonyl]amino}(phenyl)methyl](hydroxy)phos-
phoryl]methyl} pent-4-ynoic acid (2c): Yield: 95%; M.p. 163 1658C; Rf ˆ
0.38 (chloroform/methanol/acetic acid 7:2:1); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.63 1.95 (m, 1H; PCHH), 1.98 2.23
CH2indolyl), 4.27 4.58 (m, 1H; CHCONH2), 4.82 5.22 (m, 3H; PhCH2O,
PCH), 6.81 7.85 (m, 18H; Ar, indolyl, NH2), 8.07 (d, 3J(HH) ˆ 9.2 Hz, 1H;
NHCHP), 8.21 (d, 3J(HH) ˆ 9.2 Hz, 1H; NHCHCONH2); 13C NMR
(50 MHz, [D6]DMSO/1% CF3COOD): d ˆ 22.4, 27.2, 27.4 (d, J(PC) ˆ
91.0 Hz), 53.7, 55.8 (d, J(PC) ˆ 98.1 Hz), 65.9, 72.8, 81.8, 110.4, 111.3,
118.3 (d, J(PC) ˆ 11.7 Hz), 120.8, 123.6, 127.7, 128.0, 128.4, 136.1 (d,
J(PC) ˆ 8.3 Hz), 136.9, 156.1 (d, J(PC) ˆ 3.1 Hz), 172.1 (d, J(PC) ˆ 7.2 Hz),
173.2; 31P NMR (81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 42.4. HPLC:
tR ˆ 21.3 min (22.3 min for (R,R,S) diastereomer); elemental analysis calcd
(%) for C32H33N4O6P: C 63.99, H 5.54, N 9.33; found: C 63.83, H 5.39, N
9.18; ES-MS: m/z: calcd for C32H32N4O6P: 599.2; found: 599.3 [M À H]À.
ꢁ
(m, 1H; PCHH), 2.24 2.96 (m, 4H; CH2C CH, [D6]DMSO overlapping,
ꢁ
PCH2CH, C CH), 4.84 5.11 (m, 3H; PhCH2O, PCH), 7.15 7.55 (m, 10H;
Ar), 8.23 (d, J(HH) ˆ 9.7 Hz, 1H; NH); 13C NMR (50 MHz, [D6]DMSO/
3
1% CF3COOD): d ˆ 21.6, 26.8 (d, J(PC) ˆ 94.3 Hz), 54.9 (d, J(PC) ˆ
101.3 Hz), 65.5, 73.1, 80.8, 113.4, 126.2, 127.1, 128.6, 129.2, 137.3, 138.5 (d,
J(PC) ˆ 14 Hz), 156.7 (d, J(PC) ˆ 3.9 Hz), 174.2 (d, J(PC) ˆ 11.1 Hz); 31P
NMR (81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 42.1, 42.4, two diaster-
eomers (R,S)/(R,R) ˆ 3.5/1; elemental analysis calcd (%) for C21H22NO6P:
C 60.72, H 5.34, N 3.37; found: C 60.41, H 5.21, N 3.11.
(1R)-1-{[(Benzyloxy)carbonyl]amino}-2-phenylethyl[(2S)-2-({[(1S)-2-me-
thoxy-1-methyl-2-oxoethyl]amino}carbonyl)pent-4-ynyl]phosphinic acid
(3d): Preparation was performed according to the general procedure for
compounds 3a c, using the pure dipeptidic unit 2a'. After the acidic work-
up, the organic phase was dried and concentrated, and the tripeptide was
received after precipitation from diethyl ether/light petroleum (1:2). M.p.
182 1848C; Rf ˆ 0.39 (chloroform/methanol/acetic acid 7:0.5:0.5);
1H NMR (200 MHz, [D6]DMSO/1% CF3COOD): d ˆ 1.22 (d, 3J(HH) ˆ
6.7 Hz, 3H; CHCH3), 1.73 2.13 (m, 2H; PCH2), 2.33 2.60 (m, 2H;
General procedure for coupling of diacids 2a c with tryptophan amide
(3a c): To a suspension of 2a-c (1 mmol) in CH2Cl2 (20 mL per mmol)
DIPEA(3 mmol), hydrochloric l-tryptophan amide (1 mmol), HOBt
(1 mmol) and EDC ¥ HCl (4 mmol) were added. The mixture was stirred for
1.5 h at RT. Then, it was diluted with CH2Cl2 and 1m HCl was added to form
two phases. Upon addition of mineral acid the desired diastereomer (R,S,S)
was precipitated. The cloudy organic phase was washed carefully once
more with HCl and was separated. It was concentrated under vacuum and
the residue was re-dissolved in AcOEt and cooled to 08C for several hours.
The white solid, which was formed, was filtered, washed with dilute HCl,
water, and AcOEt. For compound 3b, resolution was more effectively
performed in CH2Cl2, following the same work-up. Precipitation of the
concentrated organic filtrates from diethyl ether/light petroleum (2:1)
afforded a diastereomeric mixture of the tripeptidic units essentially
enriched in the (R,R,S) isomer (e.g. for 3a de ꢀ93%, HPLC analysis).
ꢁ
CH2C CH, [D6]DMSO overlapping), 2.62 2.97 (m, 3H; PCH2CH,
ꢁ
CHHPh, C CH), 2.99 3.18 (m, 1H; CHHPh), 3.59 (s, 3H; OCH3),
3.79 4.08 (m, 1H; PCH), 4.18 4.38 (m, 1H; CHCOOCH3), 4.98 (s, 2H;
PhCH2O), 7.02 7.41 (m, 10H; Ar), 7.62 (d, 3J(HH) ˆ 9.6 Hz, 1H;
NHCHP), 8.41 (d, 3J(HH) ˆ 9.6 Hz, 1H; NHCHCOO); 13C NMR
(70 MHz, [D6]DMSO/1% CF3COOD): d ˆ 16.9, 22.3, 27.3 (d, J(PC) ˆ
87.6 Hz), 32.9, 38.1, 47.7, 51.7, 52.6 (d, J(PC) ˆ 95.9 Hz), 65.1, 72.6, 81.7,
126.2, 127.0, 127.5, 128.1, 128.3, 128.9, 137.2, 138.7 (d, J(PC) ˆ 13.6 Hz),
2090
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 2079 2094

Combinatorial Array Synthesis
2079 2094
156.0, 172.5 (d, J(PC) ˆ 7.1 Hz), 173.0; 31P NMR (81 MHz, [D6]DMSO/1%
CF3COOD): d ˆ 45.8; HPLC: tR ˆ 21.9 min; elemental analysis calcd (%)
for C26H31N2O7P: C 60.69, H 6.07, N 5.44; found: C 60.33, H 5.89, N 5.24;
ES-MS: m/z: calcd for C26H30N2O7P: 513.2; found: 513.5 [M À H]À.
(81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 45.4; HPLC: tR ˆ 28.9 min;
elemental analysis calcd (%) for C44H42N5O7P: C 67.42, H 5.40, N 8.94;
found: C 67.09, H 5.22, N 8.79; ES-MS: m/z: calcd for C44H41N5O7P: 782.3;
found: 782.3 [M À H]À.
(2S)-3-{[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}-3-oxo-
2-{[3-(3-phenoxyphenyl)isoxazol-5-yl]methyl}propyl((1R)-1-{[(benzyloxy)-
carbonyl]amino}-2-phenylethyl)phosphinic acid (5c): M.p. 215 2188C;
Rf ˆ 0.64 (chloroform/methanol/acetic acid 7:2:1); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.68 2.21 (m, 2H; PCH2), 2.52 2.85 (m,
2H; CHHPh, CHHindolyl), 2.83 3.37 (m, 5H; CH2isoxazolyl, PCH2CH,
CHHPh, CHHindolyl), 3.82 4.16 (m, 1H; PCH), 4.37 4.61 (m, 1H;
CHCONH2), 4.96 (s, 2H; PhCH2O), 6.76 (s, 1H; H of isoxazole ring),
6.85 8.58 (m, 29H; Ar, indolyl, NH, NH2); 13C NMR (50 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 27.6, 29.4, 32.9, 52.4 (d, J(PC) ˆ
105.1 Hz), 53.4, 65.2, 100.2, 110.9 (d, J(PC) ˆ 43.2 Hz), 118.4 (d, J(PC) ˆ
11.2 Hz), 120.9, 123.7, 126.3, 126.6, 126.8, 127.1, 127.5, 127.8, 128.2, 128.5,
128.7, 129.0, 133.2 (d, J(PC) ˆ 32.1 Hz), 136.1, 137.3, 138.4 (d, J(PC) ˆ
14.0 Hz), 156.2, 156.8, 157.3, 161.7, 171.7, 172.3 (d, J(PC) ˆ 9.7 Hz), 173.5;
General procedures for DCRs to tripeptidic units 3-d (5a i)
One-pot method A (for aryl-type aldoximes): The corresponding oxime
(6 equiv) was dissolved in CHCl3 (5 mL per mmol), and two drops of
pyridine were added. Then, NCS (6 equiv) was added at RT and after
10 min the resulting mixture was stirred at 458C for 3 4 h. Tripeptides 3a
c were after added (1 equiv), followed by slow addition of Et3N (7 equiv) at
the same temperature. The reaction mixture was stirred for 36 60 h at
458C. Then, it was diluted with AcOEt and washed with 1m HCl (Â2),
water, a 10% solution of NH4HCO3 (Â2; 1 mL for 30 mL AcOEt), 1m HCl
(Â2) and brine. The organic phase was dried over Na2SO4 and concentrated
under vacuum. Precipitation from diethyl ether and washings with diethyl
ether/light petroleum afforded the desired compounds (5a c,
e i).
Recovery of the starting tripeptide from the basic solution could be
performed by acidification and extraction with AcOEt. This peptide can be
re-subjected to DCRs (e.g. 5 f).
31P NMR (81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 45.3; HPLC: tR
ˆ
30.7 min; elemental analysis calcd (%) for C46H44N5O8P: C 66.90, H 5.37,
N 8.48; found: C 66.55, H 5.18, N 8.36; ES-MS: m/z: calcd for C46H43N5O8P:
824.3; found: 824.7 [M À H]À.
Two-step method B (for alkyl aldoximes e.g. product 5d): The correspond-
ing oxime (6 equiv) was dissolved in DMF (1 mL per mmol) and NCS
(6.5 equiv) was added at RT. The resulting mixture was stirred at 45 508C
for 40 min and then it was diluted with diethyl ether and extracted rapidly
with water (Â2). The organic phase was dried (Na2SO4) and concentrated
and the crude hydroximinoyl chloride was used immediately in DCRs.
Thus, this residue was dissolved in CH2Cl2 containing molecular sieves 4 ä,
and the tripeptide 3a was added (1 equiv), followed by slow addition of
Et3N (7 equiv) at RT. The reaction mixture was stirred for 48 h at RT. Then,
it was diluted with AcOEt and washed with 1m HCl (Â2), water, a 10%
solution of NH4HCO3 (Â2; 1 mL for 30 mL AcOEt), 1m HCl (Â2) and
brine. The organic phase was dried over Na2SO4 and concentrated under
vacuum. Precipitation from diethyl ether/light petroleum (0.5:9.5) and
washings with this solvent mixture afforded the desired compound (5d).
Recovery of the starting tripeptide from the basic solution could be once
more performed by acidification and extraction with AcOEt.
(2S)-3-{[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}-2-[(3-
nonylisoxazol-5-yl)methyl]-3-oxopropyl((1R)-1-{[(benzyloxy)carbonyl]a-
mino}-2-phenylethyl)phosphinic acid (5d): M.p. 76 808C; Rf ˆ 0.91
(chloroform/methanol/acetic acid 7:2:1); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 0.84 (t, 3J(HH) ˆ 6.9 Hz, 3H;
(CH2)8CH3), 1.03 1.61 (m, 17H; CH2(CH2)7CH3, CH2CH3), 1.64 2.18
(m, 2H; PCH2), 2.21 2.58 (m, 2H; CH2(CH2)7CH3, [D6]DMSO over-
lapping), 2.59 2.83 (m, 2H; CHHPh, CHHindolyl), 2.83 3.39 (m, 5H;
CH2isoxazolyl, PCH2CH, CHHPh, CHHindolyl, partially overlapped by
the multiple peak at 2.59 2.83), 3.79 4.08 (m, 1H; PCH), 4.31 4.57 (m,
1H; CHCONH2), 4.94 (s, 2H; PhCH2O), 5.81 (s, 1H; H of isoxazole ring),
3
6.65 7.81 (m, 19H; Ar, indolyl, NHCHP, NH2), 8.17 (d, J(HH) ˆ 9.2 Hz,
1H; NHCHCONH2); 13C NMR (50 MHz, [D6]DMSO/1% CF3COOD):
d ˆ 14.0, 22.1, 25.3, 25.8, 26.2, 26.6, 27.0, 27.5, 27.6, 28.0, 28.7, 28.9, 31.3, 32.8,
52.5 (d, J(PC) ˆ 105.3 Hz), 53.5, 65.2, 101.6, 110.5, 111.3, 112.3, 118.1, 118.3
(d, J(PC) ˆ 11.0 Hz), 120.8, 123.5, 123.8, 126.2, 127.0, 127.4, 127.5, 128.2,
128.3, 129.0, 136.1, 137.2, 138.4 (d, J(PC) ˆ 14.4 Hz), 156.1 (d, J(PC) ˆ
3.4 Hz), 163.4, 170.1, 172.2 (d, J(PC) ˆ 9.1 Hz), 173.4; 31P NMR (81 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 45.4; HPLC: tR ˆ 35.8 min; elemental
analysis calcd (%) for C43H54N5O7P: C 65.88, H 6.94, N 8.93; found: C
65.72, H 6.88, N 8.79; ES-MS: m/z: calcd for C43H53N5O7P: 782.4; found:
782.3 [M À H]À.
3-{[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}-3-oxo-2-
[(3-phenylisoxazol-5-yl)methyl]propyl((1R)-1-{[(benzyloxy)carbonyl]ami-
no}-2-phenylethyl)phosphinic acid (5a): M.p. 165 1698C; Rf ˆ 0.47
(chloroform/methanol/acetic acid 7:2:1); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.62 2.21 (m, 2H; PCH2), 2.52 2.82
(m, 2H; CHHPh, CHHindolyl), 2.84 3.39 (m, 5H; CH2isoxazolyl,
PCH2CH, CHHPh, CHHindolyl), 3.82 4.17 (m, 1H; PCH), 4.33 4.57
(m, 1H; CHCONH2), 4.92 (s, 2H; PhCH2O), 6.39 (R,R,S) and 6.57 (R,S,S)
(s, 1H; H of isoxazole ring), 6.62 7.92 (m, 24H; Ar, indolyl, NHCHP,
NH2), 8.23 (R,S,S) and 8.49 (R,R,S) (d, 3J(HH) ˆ 9.2 Hz, 1H;
NHCHCONH2); 13C NMR (50 MHz, [D6]DMSO/1% CF3COOD): d ˆ
27.6, 29.6, 32.8, 52.4 (d, J(PC) ˆ 105.3 Hz), 53.5, 65.2, 100.1, 100.6,
110.9 (d, J(PC) ˆ 43.1 Hz), 111.6 (d, J(PC) ˆ 63.6 Hz), 118.0, 118.3,
118.5, 120.9, 123.5, 126.2, 126.9, 127.0, 127.4, 127.5, 128.2, 128.5, 128.8,
129.0, 130.0, 136.1, 137.1, 138.3 (d, J(PC) ˆ 14.0 Hz), 156.0, 161.7, 170.9,
171.6, 172.2 (d, J(PC) ˆ 9.8 Hz), 173.5, 173.8; 31P NMR (81 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 45.4 for (R,S,S) diastereomer, 46.2 for
(R,R,S) diastereomer; HPLC: tR ˆ 26.0 min (R,S,S)/27.1 min (R,R,S);
elemental analysis calcd (%) for C40H40N5O7P: C 65.48, H 5.49, N 9.54;
found: C 65.22, H 5.33, N 9.29; ES-MS: m/z: calcd for C40H39N5O7P: 732.3;
found: 732.5 [M À H]À.
(2S)-3-{[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}-2-{[3-
(2-naphthyl)isoxazol-5-yl]methyl}-3-oxopropyl((1R)-1-{[(benzyloxy)car-
bonyl]amino}-2-phenylethyl)phosphinic acid (5b): M.p. 208 2108C; Rf ˆ
0.60 (chloroform/methanol/acetic acid 7:2:1); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.68 2.22 (m, 2H; PCH2), 2.52 2.83
(m, 2H; CHHPh, CHHindolyl), 2.85 3.39 (m, 5H; CH2isoxazolyl,
PCH2CH, CHHPh, CHHindolyl), 3.82 4.16 (m, 1H; PCH), 4.37 4.61
(m, 1H; CHCONH2), 4.94 (s, 2H; PhCH2O), 6.78 (s, 1H; H of isoxazole
ring), 6.82 8.43 (m, 27H; Ar, indolyl, NH, NH2); 13C NMR (50 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 27.6, 29.5, 32.9, 52.2 (d, J(PC) ˆ
104.8 Hz), 53.5, 65.2, 100.3, 110.9 (d, J(PC) ˆ 43.2 Hz), 118.3 (d, J(PC) ˆ
11.3 Hz), 120.9, 123.7, 126.3, 126.4, 126.8, 127.0, 127.5, 127.8, 128.2, 128.5,
128.7, 129.0, 133.2 (d, J(PC) ˆ 32.2 Hz), 136.1, 137.1, 138.3 (d, J(PC) ˆ
14.0 Hz), 156.1, 161.8, 171.7, 172.2 (d, J(PC) ˆ 9.8 Hz), 173.5; 31P NMR
(1R)-1-{[(Benzyloxy)carbonyl]amino}-2-phenylethyl{3-{[(1S)-2-methoxy-
1-methyl-2-oxoethyl]amino}-3-oxo-2-[(3-phenylisoxazol-5-yl)methyl]pro-
pyl}phosphinic acid (5e): M.p. 145 1488C; Rf ˆ 0.41/0.30 (two diaster-
eomers) (chloroform/methanol/acetic acid 7:1:0.5); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.18 (R,R,S) and 1.27 (R,S,S) (d,
3J(HH) ˆ 7.2 Hz, 3H; CHCH3), 1.67 2.17 (m, 2H; PCH2), 2.59 2.93 (m,
2H; CHHisoxazolyl, CHHPh), 2.97 3.46 (m, 3H; PCH2CH, CHHPh,
CHHisoxazolyl), 3.51 (s, 3H; OCH3), 3.77 4.11 (m, 1H; PCH), 4.17 4.38
(m, 1H; CHCO2CH3), 4.84/4.98 (s, 2H; PhCH2O), 6.75 (R,R,S) and 6.80
(R,S,S) (s, 1H; H of isoxazole ring), 6.87 7.88 (m, 16H; Ar, NHCHP), 8.41
(R,R,S) and 8.56 (R,S,S) (d, 3J(HH) ˆ 9.2 Hz, 1H; NHCHCOO); 13C NMR
(70 MHz, [D6]DMSO/1% CF3COOD): d ˆ 16.8, 22.3, 27.4 (d, J(PC) ˆ
88.8 Hz), 32.9, 38.1, 47.6, 51.7, 52.8 (d, J(PC) ˆ 96.1 Hz), 65.1, 100.2, 100.8,
126.2, 127.1, 127.3, 128.1, 128.3, 128.8, 137.2, 138.6 (d, J(PC) ˆ 13.8 Hz),
156.2, 161.6, 170.8, 171.2, 172.5 (d, J(PC) ˆ 7.1 Hz), 173.3; 31P NMR
(81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 45.4 for (R,S,S) diastereomer,
45.8 for (R,R,S) diastereomer; HPLC: tR ˆ 28.3 min (R,S,S)/29.4 min
(R,R,S); elemental analysis calcd (%) for C33H36N3O8P: C 62.55, H 5.73,
N 6.63; found: C 62.67, H 5.88, N 6.72; ES-MS: m/z: calcd for C33H35N3O8P:
632.2; found: 632.4 [M À H]À.
(1R)-1-{[(Benzyloxy)carbonyl]amino}-2-phenylethyl((2S)-3-{[(1S)-2-me-
thoxy-1-methyl-2-oxoethyl]amino}-2-{[3-(4-nitrophenyl)isoxazol-5-yl]-
methyl}-3-oxopropyl)phosphinic acid (5 f): M.p. 219 2218C; Rf ˆ 0.44
(chloroform/methanol/acetic acid 7:2:1); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.26 (d, 3J(HH) ˆ 7.4 Hz, 3H; CHCH3),
1.68 2.18 (m, 2H; PCH2), 2.57 2.95 (m, 2H; CHHisoxazolyl, CHHPh),
2.99 3.38 (m, 3H; PCH2CH, CHHPh, CHHisoxazolyl), 3.51 (s, 3H;
Chem. Eur. J. 2003, 9, 2079 2094
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2091

A. Yiotakis et al.
FULL PAPER
OCH3), 3.77 4.08 (m, 1H; PCH), 4.14 4.36 (m, 1H; CHCO2CH3), 4.93 (s,
2H; PhCH2O), 6.97 (s, 1H; H of isoxazole ring), 6.90 7.38, 7.62 7.7.75,
8.04 8.20, 8.33 8.44 (m, 15H; Ar, NHCHP), 8.58 (d, 3J(HH) ˆ 7.2 Hz,
1H; NHCHCOO); 13C NMR (70 MHz, [D6]DMSO/1% CF3COOD): d ˆ
16.8, 22.3, 27.5 (d, J(PC) ˆ 89.1 Hz), 32.9, 38.2, 47.3, 51.6, 52.8 (d, J(PC) ˆ
96.2 Hz), 65.3, 100.4, 126.2, 127.2, 127.3, 128.2, 128.3, 128.7, 137.2, 138.4 (d,
J(PC) ˆ 13.9 Hz), 147.9, 156.2, 161.7, 171.2, 172.5 (d, J(PC) ˆ 9.4 Hz), 173.3;
General procedures for preparation of tripeptides of type 9 on solid phase
(pins)
Fmoc deprotection: Fmoc-Rink amide derivatized pins (L-series pins,
loading: 14 mmol per pin) were treated with a 20% piperidine/DMF
solution (0.5 mL per pin, 2 Â 30 min, RT). Each time, the solvent was
decanted, and the pins were washed with DMF (2 Â 5 min, 1 mL per pin)
and CH2Cl2 (2 Â 5 min, 1 mL per pin) and dried in vacuo.
Fmoc-tryptophan coupling: The deprotected pins were treated with a
preactivated (10 min) mixture of Fmoc-tryptophan (3 equiv per pin,
0.12m), DIC (3 equiv per pin, 0.12m) and HOBt (3 equiv per pin, 0.12m)
in a CH2Cl2/DMF (6:1 v/v) solution for 4 5 h at RT. Then, the solution was
decanted and the pins were washed with CH2Cl2 (2 Â 5 min, 1 mL per pin),
DMF (2 Â 5 min, 1 mL per pin), and CH2Cl2 (2 Â 5 min, 1 mL per pin) and
briefly air-dried to give 6a (complete coupling was monitored by a negative
Kaiser test). Fmoc-deprotection of 6a was performed applying the above
described deprotection method to afford 6b.
31P NMR (81 MHz, [D6]DMSO/1% CF3COOD): d ˆ 45.2; HPLC: tR
ˆ
27.1 min; elemental analysis calcd (%) for C33H35N4O10P: C 58.41, H 5.20,
N
8.26; found: C 58.32, H 5.07, N 8.34; ES-MS: m/z: calcd for
C33H34N4O10P: 677.2; found: 677.2 [M À H]À.
(2S)-3-{[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}-2-[(3-
[1,1'-biphenyl]-4-ylisoxazol-5-yl)methyl]-3-oxopropyl((1R)-1-{[(benzyloxy)-
carbonyl]amino}ethyl)phosphinic acid (5g): Part of 5g was recovered from
the basic solution and purified by column chromatography (chloroform/
methanol/acetic acid 7:0.8:0.5). Yield given in Table 3 is after combination
with the isolated product by precipitation. M.p. 165 1688C; Rf ˆ 0.52
(chloroform/methanol/acetic acid 7:1:0.5); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.21 (dd, 3J(HH) ˆ 7.3 Hz, 3J(PH) ˆ
14.6 Hz, 3H; CHCH3), 1.63 2.08 (m, 2H; PCH2), 2.39 2.45 (m, 1H;
CHHindolyl, [D6]DMSO/overlapping), 2.89 3.23 (m, 4H; PCH2CH,
CHHindolyl, CH2isoxazolyl), 3.64 3.83 (m, 1H; PCH), 4.37 4.56 (m,
1H; CHCONH2), 5.01 (s, 2H; PhCH2O), 6.59 (s, 1H; H of isoxazole ring),
Protocol for P-unprotected dipeptidic block (2a') coupling: The dipeptidic
block 2a' (1.5 equiv per pin, 0.08m) was added to pins in CH2Cl2 (0.25 mL
per pin) containing DIPEA(1.5 equiv per pin). HOBt (1.5 equiv per pin,
0.08m), EDC ¥ HCl (4 equiv per pin, 0.22m) and DIPEA(1 equiv per pin,
0.14m overall concentration) were added sequentially to the pins solution.
The reaction mixture was shaken from time to time and after 2 h the
coupling was stopped by decanting the solution. The pins were washed with
CH2Cl2 (2 Â 5 min, 1 mL per pin), DMF (2 Â 5 min, 1 mL per pin), and
CH2Cl2 (2 Â 5 min, 1 mL per pin) and briefly air-dried. The coupling was
performed once more under the same conditions to afford 7 (Kaiser test
and HPLC analysis were used to monitor the progress of the reaction and
racemization). If racemization is not a problem (e.g. use of 2a), the
coupling can be performed only once, overnight.
3
6.83 7.89 (m, 28H; Ar, indolyl, NHCHP, NH2), 8.26 (d, J(HH) ˆ 8.4 Hz,
1H; NHCHCONH2); 13C NMR (70 MHz, [D6]DMSO/1% CF3COOD):
d ˆ 14.3, 22.3 (d, J(PC) ˆ 9.4 Hz), 27.1 (d, J(PC) ˆ 90.6 Hz), 27.2, 46.1 (d,
J(PC) ˆ 103.6 Hz), 53.5, 65.3, 100.2, 110.4, 111.3, 118.2 (d, J(PC) ˆ 15.6 Hz),
120.8, 123.8, 126.3, 126.7, 127.4, 127.7, 127.8, 128.3, 128.5, 128.8, 129.0, 136.1,
137.0, 138.8, 139.1, 139.8, 140.2, 142.2, 155.9 (d, J(PC) ˆ 4.6 Hz), 161.8,
171.6, 172.1 (d, J(PC) ˆ 8.4 Hz), 173.3; 31P NMR (81 MHz, [D6]DMSO/1%
CF3COOD): d ˆ 46.2; HPLC: tR ˆ 27.3 min; elemental analysis calcd (%)
for C40H40N5O7P: C 65.48, H 5.49, N 9.54; found: C 65.23, H 5.17, N 9.28;
ES-MS: m/z: calcd for C40H39N5O7P: 732.3; found: 732.5 [M À H]À.
General procedure for DCRs to tripeptides of type 7 (Following the
optimized conditions described in Figure 6): The corresponding oxime
(10 equiv per pin, 0.56m) was dissolved in CHCl3 (0.25 mL per pin), and
one drop of pyridine was added (use of chemically resistant glass tubes of
the desired dimensions). Then, NCS (10 equiv per pin, 0.56m) was added at
RT and after 10 min the resulting mixture was heated at 458C for 4 h, with
shaking from time to time. Tripeptide 7 was after added (1 equiv per pin),
followed by slow addition of Et3N (11 equiv per pin) at the same
temperature. The reaction mixture was left for 48 h at 458C, with shaking
occasionally. The pins were washed with CH2Cl2 (2 Â 5 min, 1 mL per pin),
DMF (2 Â 5 min, 1 mL per pin), MeOH (1 Â 5 min, 1 mL per pin), DMSO
(1 Â 5 min, 1 mL per pin, 608C) and CH2Cl2 (2 Â 5 min, 1 mL per pin) and
briefly air-dried. DCRs were repeated twice (or three) more as above to
afford 8. HPLC analysis of cleaved products was used to monitor the
progress of the reaction. The final isoxazole-containing phosphinic
tripeptidic array of type 9 was obtained after treatment of 8 with a mixture
of TFA/H2O/TIS (95:2.5:2.5, 0.5 mL per pin) for 1 h at RT. After acidic
cleavage and removal of the pins from the reaction mixture, the volatiles
were removed by an argon stream. The oily residue was precipitated with
cold diethyl ether and the mixture was centrifuged. The organic phase was
carefully decanted and the residue was re-subjected to the same treatment
once more. The off-white solid (product 9) was analyzed by analytical RP-
HPLC and ES-MS (see Table 4). Further purification and separation of
diastereomers, if necessary, was performed by semipreparative RP-HPLC.
(2S)-3-{[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}-2-{[3-
(4-fluorophenyl)isoxazol-5-yl]methyl}-3-oxopropyl[(R)-{[(benzyloxy)car-
bonyl]amino}(phenyl)methyl]phosphinic acid (5h): M.p. 242 2448C; Rf ˆ
0.41 (chloroform/methanol/acetic acid 7:2:1); 1H NMR (200 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 1.62 2.18 (m, 2H; PCH2), 2.23 3.36
(m, 5H; CH2isoxazolyl, PCH2CH, CH2indolyl, [D6]DMSO overlapping),
4.28 4.56 (m, 1H; CHCONH2), 4.82 5.18 (m, 3H; PhCH2O, PCH), 6.54
(s, 1H; H of isoxazole ring), 6.95 8.56 (m, 29H; Ar, indolyl, NH, NH2);
13C NMR (50 MHz, [D6]DMSO/1% CF3COOD): d ˆ 27.5, 27.9 (d, J(PC) ˆ
91.1 Hz), 53.6, 55.8 (d, J(PC) ˆ 98.1 Hz), 65.9, 100.2, 110.4, 111.4, 116.1 (d,
J ˆ 21.6 Hz), 118.3 (d, J ˆ 11.8 Hz), 120.8, 123.6, 127.2, 127.8, 128.1, 128.8,
128.9, 136.1, 136.8, 156.2 (d, J(PC) ˆ 3.8 Hz), 160.9, 163.1 (d, J(FC) ˆ
247.3 Hz), 171.5, 172.2 (d, J(PC) ˆ 7.6 Hz), 173.4; 31P NMR (81 MHz,
[D6]DMSO/1% CF3COOD): d ˆ 41.9; HPLC: tR ˆ 25.7 min; elemental
analysis calcd (%) for C39H37FN5O7P: C 63.50, H 5.06, N 9.49; found: C
63.37, H 4.96, N 9.34; ES-MS: m/z: calcd for C39H36FN5O7P: 736.2; found:
736.3 [M À H]À.
3-{[(1S)-2-Amino-1-(1H-indol-3-ylmethyl)-2-oxoethyl]amino}-2-{[3-(2-fur-
yl)isoxazol-5-yl]methyl}-3-oxopropyl[(R)-{[(benzyloxy)carbonyl]amino}
(phenyl)methyl]phosphinic acid (5i): M.p. 190 1958C (decomp); Rf ˆ
0.51/0.54 (two diastereomers) (chloroform/methanol/acetic acid 7:2:1);
1H NMR (200 MHz, [D6]DMSO/1% CF3COOD): d ˆ 1.59 2.08 (m, 2H;
PCH2), 2.21 3.40 (m, 5H; CH2isoxazolyl, PCH2CH, CH2indolyl,
[D6]DMSO overlapping), 4.25 4.56 (m, 1H; CHCONH2), 4.86 5.20 (m,
3H; PhCH2O, PCH), 6.35/6.45 (s, 1H; H of isoxazole ring), 6.65 8.45 (m,
23H; Ar, furyl, indolyl, NH, NH2); 13C NMR (50 MHz, [D6]DMSO/1%
CF3COOD): d ˆ 27.3, 27.5 (d, J(PC) ˆ 90.8 Hz), 53.7, 55.8 (d, J(PC) ˆ
98.1 Hz), 65.4, 101.2, 102.3, 110.4, 111.3, 112.8, 114.2, 118.3 (d, J(PC) ˆ
11.7 Hz), 118.6, 120.8, 123.6, 126.3, 126.7, 127.2, 127.5, 127.7, 128.1, 128.4,
136.1 (d, J(PC) ˆ 8.5 Hz), 136.6, 137.2, 138.4 (d, J(PC) ˆ 13.8 Hz), 144.4,
145.1, 148.6, 156.1 (d, J(PC) ˆ 3.8 Hz), 162.0, 170.6, 171.3, 172.1 (d, J(PC) ˆ
7.6 Hz), 173.2, 173.9; 31P NMR (81 MHz, [D6]DMSO/1% CF3COOD): d ˆ
41.8, 42.4 (two diastereomers); HPLC: tR ˆ 23.3 min (R,S,S)/24.1 min
(R,R,S); elemental analysis calcd (%) for C37H36N5O8P: C 62.62, H 5.11,
N 9.87; found: C 62.33, H 4.86, N 9.56; ES-MS: m/z: calcd for C37H35N5O8P:
708.2; found: 708.4 [M À H]À.
Acknowledgement
This work was supported by funds from the National and Kapodistrian
University of Athens, Special Account for Research Grants. We are
grateful to Alexander S. Onassis Foundation for a generous scholarship (to
A.M.).
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Received: September 26, 2002 [F4456]
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