Design, synthesis and biological evaluation of nonpeptide integrin antagonists

Article abstract of DOI:10.1016/S0968-0896(98)00090-X

Recent studies demonstrated that peptide and antibody antagonists of integrin α(v)β₃ block angiogenesis and tumor growth. In this article, the design, synthesis and biological evaluation of a series of nitroaryl ether-based, nonpeptide mimetics are described. The design of these compounds was based on Merck's arylether/α-aminoacid/guanidine framework and incorporates a novel nitroaryl system. The synthesized mimetics were tested against a variety of integrins (α(v)β₃, α(IIb)β₃, and α(v)β₅) in order to determine their binding selectivity and ability to inhibit cell adhesion. Selected compounds were also tested for their ability to inhibit angiogenesis in vivo in the CAM (chick chorioallantoic membrane) assay. From the generated compound library, compounds 16 and 19 proved to be potent and selective inhibitors of α(IIb)β₃ (IC₅₀=14nM) whereas compound 11 showed excellent in vivo inhibition of angiogenesis (at 30μg/embryo). Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00090-X

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1185±1208
Design, Synthesis and Biological Evaluation of Nonpeptide
Integrin Antagonists
K. C. Nicolaou,a, c,* John I. Trujillo,a Bernd Jandeleit,a Kelly Chibale,a
M. Rosenfeld,b B. Diefenbach,d D. A. Chereshb and S. L. Goodmand
aDepartment of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037, USA
bDepartments of Immunology, and Vascular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla,
CA 92037, USA
cDepartment of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
dMerck KGaA Preclinical Research, Frankfurter Strasse 250, D-64271 Darmstadt, Germany
Received 20 March 1998; accepted 1 May 1998
AbstractÐRecent studies demonstrated that peptide and antibody antagonists of integrin avb3 block angiogenesis and
tumor growth. In this article, the design, synthesis and biological evaluation of a series of nitroaryl ether-based, non-
peptide mimetics are described. The design of these compounds was based on Merck's arylether/a-aminoacid/guani-
dine framework and incorporates a novel nitroaryl system. The synthesized mimetics were tested against a variety of
integrins (avb3, aIIbb3, and avb5) in order to determine their binding selectivity and ability to inhibit cell adhesion.
Selected compounds were also tested for their ability to inhibit angiogenesis in vivo in the CAM (chick chorioallantoic
membrane) assay. From the generated compound library, compounds 16 and 19 proved to be potent and selective
inhibitors of aIIbb3 (IC50=14 nM) whereas compound 11 showed excellent in vivo inhibition of angiogenesis (at 30 mg/
embryo). # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
platelet aggregation and several groups have already
disclosed the design and synthesis of potent binders with
peptide and nonpeptidal structures.2 Within the context
of angiogenesis, the functions of avb3 and avb5 have
been shown to be vital.3 Cheresh and co-workers have
shown that in vivo inhibition of binding of these integ-
rins to their native ligands by antibodies or cyclic pep-
tides interferes with angiogenesis and induces tumor
regression.4 In addition to its relevance to angiogenesis,
avb3 has also been shown to play a role in mediating
adhesion of osteoclasts to the bone matrix5 and in the
migration of vascular smooth muscle cells.6 Antagonists
of avb3 are, therefore, envisioned as potential thera-
peutic agents for the treatment of numerous disease
states such as diabetic retinopathy,7 cancer,4 osteo-
porosis5 and restenosis.6
The integrins are a class of cell surface proteins that
facilitate cell±cell and cell±matrix adhesion.1 These
important biological targets are membrane bound, het-
erodimeric glycoproteins made up of an a-subunit and a
b-subunit. The relative anity and speci®city for ligand
binding is determined by the unique combination of the
di€erent a- and b-subunits. Of the members of this
family of receptors, aIIbb3, a5b1, avb3 and avb5 are
extensively studied. A number of known natural ligands
to these integrins, such as ®bronectin (binds to a5b1 and
avb3), ®brinogen (binds to aIIbb3 and avb3) and vitro-
nectin (binds to avb3 and avb5), contain common pep-
tide sequence within their native sequence, which are
recognized by several integrins. The aIIbb3 integrin was
shown to be an excellent target for the inhibition of
The ®rst small molecule antagonists of avb3 were repor-
ted by Kessler et al.8 (e.g. 1, Fig. 1). Subsequently,
groups from Dupont-Merck9a (e.g. 2, Fig. 1) and
*Corresponding author.
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00090-X

1186
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
Figure 1. Selected structures of avb3 antagonists.
SmithKline Beecham9b (SKB) (e.g. 3, Fig. 1) pub-
lished their results in the ®eld. In addition, cyclic
peptides such as 4 and 5 (Fig. 1) were synthesized and
shown to be active by Burgess et al.9c and Tran et al.,9d
respectively.
Results and Discussion
Design and retrosynthetic analysis
Figure 3 shows the targeted compounds (10±22) for the
present study. Amongst the considerations that led to
their design were: (a) the Merck10a ®ndings pointing to
the importance of the guanidine/aryl sulfonamide func-
tionalities; and (b) the facile entry into such structures
from o-nitro-aryl¯uorides as shown in Fig. 4. The
designed molecules fall within the general structure I
(Fig. 4), which can be derived by coupling the central
nitro¯uoroaromatic system II with fragments III
(nucleophile) and IV (aminoacid component).
More recently a number of groups reported their results
with high anity ligands for avb3 possessing structures
signi®cantly deviating from classical peptide frame-
works (e.g. 6±9, Fig. 2). These structures contain a
central sca€old (e.g. benzene,10a benzodiazepine-
type,10b,d or urea10c backbone) onto which appendages
carrying carboxylate and guanidino groups are
attached. In our group we have previously utilized car-
bohydrate frameworks11 as sca€olds for the design and
synthesis of potential avb3 antagonsists.12 In this article
we describe the design, chemical synthesis and biological
Chemical synthesis
For the synthesis of compounds 10±22 (Fig. 3), the
amino acid derivatives 26, 29a, and 29b were required.
These intermediates were obtained from l-asparagine
evaluation of a series of nitroaryl-based integrin
antagonists and their use as anti-angiogenic agents.

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1187
Figure 2. Selected nonpeptide mimetics with high anity for avb3.
(23) as outlined in Scheme 1. Thus, conversion of 23
to its Boc derivative (24, 88%) under standard con-
ditions was followed by benzyl ester formation
(Cs2CO3±BnBr)13a to a€ord 25 (88% yield). Conversion
of the amide to the amine by a decarboxylative
rearrangement with PhI(OCOCF3)2 furnished derivative
26 in 41% yield.13b,c The sulfonamides 29a and 29b were
prepared by sulfonylation of the amino group of 23 to
a€ord 27a and 27b, followed by Ho€mann rearrange-
ment and esteri®cation of the resulting aminoacids (28a
and 28b) with isobutylene (Scheme 1). Details and yields
for these transformations can be found in Scheme 1.14
For the synthesis of the sulfonamide compounds 11±13,
the common intermediate 34 was deprotected (TFA,
84%) and the liberated amine (37) was reacted with the
appropriate sulfonyl chloride to a€ord compounds 38a
(78%) and 38b (57%). Reduction of the azide function-
ality in 38a and 38b with Ph3P±H2O, again resulted in a
mixture of the corresponding primary amine (39a and
39b) and its rearranged primary alcohol (41a and 41b) in
80% total yield. Basic hydrolysis of 39a and 39b resulted
in the formation of the corresponding carboxylic acids
(40a and 40b, 93±99% yield), guanylation of which as
described above furnished the desired compounds 11
and 13 (13±15% yield, unoptimized) respectively. Simi-
larly, hydrolysis of 41a (LiOH, 96% yield) followed by
guanylation furnished compound 12 in low yield, via
compound 42a.
Scheme 2 summarizes the initial approach to com-
pounds 10±13. Thus, 4-¯uoro-o-nitrobenzoic acid (30)
was converted to its methyl ester (31, 98%) by treatment
with trimethylorthoacetate15 at 80 ꢀC, and thence reac-
ted with N3(CH2)2OTBS in DMF16 in the presence of
catalytic amounts of TBAF resulting in the formation of
compound 32 (73%). Saponi®cation of 32 (LiOH, 99%
yield) furnished carboxylic acid 33 which was condensed
with building block 26 in the presence of DCC and
4-DMAP to give key intermediate 34 (82% yield). For the
synthesis of 10, compound 34 was reduced with Ph3P in
the presence of H2O, to a€ord amine 35a, together with
the rearranged product 35b (80% combined yield) in
which the side chain heteroatoms have interchanged
positions (via an internal nucleophilic attack, see struc-
ture 35a).17 On standing at ambient temperature, pri-
mary amine 35a underwent quantitative conversion to
primary alcohol 35b. However, it was possible to rapidly
manipulate the compound through basic hydrolysis
The rearrangement observed during the reduction of the
side-chain azido group led us to explore an alternative
strategy for the construction of the targeted nitroaryl
ether compounds. According to the new plan, a nucleo-
philic species containing a fully protected guanidine
moiety was to be employed in the displacement of the
¯uoride from the central nitroaryl system. To this end,
the nucleophiles 51±5619±21 were prepared from readily
available starting materials and by standard chemistry
as outlined in Scheme 3. The incorporation of these
fragments into the main-frame of the molecule via
nucleophilic aromatic substitution,16,17 and the synthesis
of the ®nal targets are shown in Scheme 4 (11, 14±19)
and Scheme 5 (20 and 21). Thus, the acid chloride 57
(derived from carboxylic acid 30) was coupled with
amines 29a and 29b in the presence of Et3N to a€ord
amides 58 (98%) and 59 (96%), respectively. Coupling
of 58 with nucleophile 51 was e€ected in the presence of
(LiOH) and guanylation (1H-pyrazole-1-carbox-
18
.
amidine HCl) and obtain the targeted compound 10,
albeit in low yield (15% after RP-HPLC puri®cation).

1188
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
Figure 3. Targeted nitroaryl ethers (10±21) and benzimidazole 22.
NaH in DMF to a€ord product 60 in 66% yield. Simi-
larly, 63 was obtained by coupling 59 with 51 (69%
yield). The amino compounds 61 and 64 were obtained
from 58 and 59 in 73 and 99% yield, respectively, by
reaction with amine 53 in DMF at ambient tempera-
ture. Thioether 62 was obtained by exposure of 58 to
thiol 54 and NaH (DMF, 25 ꢀC, 23% yield). Treatment
of compounds 60±64 with TFA in CH2Cl2 at room
temperature resulted in concomitant deprotection of
both the guanidine and carboxyl groups in excellent
yield (90±99%, after RP-HPLC puri®cation). The
piperazine compounds 16 and 19 were prepared in a
similar fashion from 58 and 59 respectively by ®rst dis-
placing the ¯uoride with nucleophile 52, followed by
TFA-induced deprotection of the resulting derivatives
65 and 66 as summarized in Scheme 4.

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1189
Figure 4. General structures of nitroaryl ether peptide mimetics and retrosynthetic analysis.
Scheme 1. Synthesis of amino esters 26, 29a, and 29b. Reagents and conditions: (a) 1.1 equiv of Boc2O, 1.0 equiv of Na2CO3, 1,4-
dioxane, H2O, 25 ꢀC, 88%; (b. i.) 20% aq solution of Cs2CO3, H2O:MeOH (1:2.5), 25 ꢀC, 4 h, 100%; (ii) 1.1 equiv of BnBr, DMF,
25 ꢀC, 14 h, 88%; (c) 1.5 equiv of PhI(OCOCF3)2, DMF:H2O (1:1), 2.0 equiv of pyridine, 25 ꢀC, 3.5 h, 41%; (d) 1.1 equiv of ArSO2Cl, 2.25
equiv of NaOH, dioxane:H2O (1:2), 0!25 ꢀC, 3 h, [71% for 27a, 66% for 27b]; (e) 1.3 equiv of Br2, 9.2 equiv of NaOH, H2O,
0!90 ꢀC, [75% for 28a, 81% for 28b]; (f) isobutylene, 2.8 equiv of concd H2SO4, DME, 78!25 ꢀC, 48 h, [55% for 29a, 51% for 29b].
DME=dimethoxyethane; DMF=dimethylformamide; 2-Naph=2-naphthyl.
The synthesis of compounds 20 and 21 is shown in
Scheme 5. Thus, reaction of 58 with 55 in the presence
of Et3N at 25 ꢀC in DMF resulted in the formation of
compound 67 (92% yield) which was exposed to
TFA:CH2Cl2 (1:1) at 25 ꢀC to a€ord targeted benzimid-
azole 20 (97% yield). In a similar fashion, compound 21
was prepared via the intermediacy of 68 by reaction of
58 with 56 (Et3N, DMF, 25 ꢀC) followed by deprotec-
tion (83% after RP-HPLC puri®cation).
Finally, the preparation of compound 22 is shown in
Scheme 6. Thus, key intermediate 57 was reacted with
ammonia in DMF to a€ord nitroaniline 69 in 93%
yield. Reduction of 69 with H2 in the presence of 10%

1190
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
Scheme 2. Synthesis of compounds 10±13. Reagents and conꢀditions: (a) 5.0 equiv of MeC(OMe)3, PhMe, 80 ꢀC, 8 h, 98%; (b) 1.1 equiv
ꢀ
.
of N3(CH2)2OTBS, 0.1 equiv of TBAF, 4 A MS, DMF, 25 C, 4 h, 73%; (c) 2.0 equiv of LiOH H2O, dioxane:H2O (3:1), 25 C, 4 h
99%; (d) 1.0 equiv of DCC, 0.2 equiv of 4-DMAP, CH2CL2, 25 ꢀC, 4 h, 82%; (e) 50% TFA in CH2Cl2, 25 ꢀC, 2 h, 84%; (f) 1.1 equiv
of PhSO2Cl or 1-NaphSO2Cl, 1.3 equiv of i-Pr2NEt, CH2Cl2, 25 ꢀC, 4 h, 38a (78%), or 38b (57%); (g) 2.0 equiv of Ph3P, 44 equiv of
ꢀ
.
H2O, THF, 25 C, 12 h, 80%, ca. 1:1 of 35a:35b; 80%, ca. 1:1 of 39a:41a: 81%, ca. 1:1 of 39b:41b; (h) 2.0 equiv of LiOH H2O,
ꢀ
.
THF:H2O (3:1), 25 C, 4 h, 93±99% for 36ab, 40ab, 42a; (i) 1.1 equiv of 1H-pyrazole-1-carboxamidine HCl, 1.1 equiv of i-Pr2NEt,
DMF, 25 ꢀC, 16 h, 13±15% for 10, 11, 13: 50 ꢀC, 16 h, 5% for 12, after RP-HPLC. TFA=tri¯uoroacetic acid; TBAF=tetra-n-butyl-
ammonium ¯uoride; DCC=1,3-dicyclohexylcarbodiimide.
Pd/C catalyst in MeOH led to 1,2-diamine 70 (90%),
which reacted with phenylisothiocyanate in EtOH to
a€ord thiourea 71 (69% yield). Treatment of 71 with
HgCl2 and Et3N in DMF at ambient temperature gave
guanidine 72 in 81% yield. Cleavage of the tert-butyl
ester in 72 with TFA in CH2Cl2 then led to the targeted
compound 22 (88% yield, after RP-HPLC puri®cation).
Biological evaluation
In integrin inhibition assays, the nitroaryl ether
mimetics revealed high activity and selectivity (Table 1).
Compound 1 had an IC50 of 3 nM on avb3, 1.8 mM
on avb5 and 6.4 mM on aIIbb3. The nitroaryl com-
pounds 10±21 (Fig. 3) were all essentially inactive on

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1191
Scheme 3. Synthesis of guanidine derivatives 51±56. Reagents and conditions: (a) 1.0 equiv of BtBMTP, 2.0 equiv of Et3N, 1.0 equiv of
HgCl2, DMF, 25 ꢀC, 4 h, 98%; (b) 1.0 equiv of BtBMTP, DMF, 25 ꢀC, 14 h, 95%; (c) 1.0 equiv of BtBCT, 2.0 equiv of Et3N, DMF,
25 dC, 14 h, 60%; (d) 0.2 equiv of BtBMTP, 0.4 equiv of Et3N, 0.2 equiv of HgCl2, DMF, 25 ꢀC, 4 h, 51%; (e) 0.66 equiv of o-
ꢀ
.
NH2C6H4NH2, 5.5 N aq HCl, re¯ux, 24 h, 73%; (f) 1.0 equiv of DmPD HBr, i-Pr2NEt, DMF, 25 C, 11 h, 51%. Boc=tert-butoxy-
carbonyl; BtBMTP=1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea;
BtBCT=N,N0-bis-tert-butoxycarbonylthiourea;
DmPD HBr-2-(3,5-dimethylpyrazolyl)-4,5-dehydroimidazole hydrobromide.
.
avb5, none having an IC50 of below 1 mM. Some were
speci®c inhibitors of avb3 (e.g. compound 20), others
were mixed inhibitors of both avb3 and aIIbb3 (e.g.
compounds 15 and 18) or aIIbb3-speci®c inhibitors
(e.g. compound 19). The degree of selectivity for avb3
over aIIbb3 was up to two orders of magnitude
(compound 20), and thus comparable with selective
peptidic inhibitors of avb3 (e.g. compound 1), while
monospeci®c aIIbb3 inhibitors were also found (com-
pound 12). The activity of the compounds was high,
adhesion of M21-L-IIb cells to ®brinogen did not reach
an IC50 value at 100 mM inhibitor.
Compounds 16 and 19 speci®cally inhibited aIIbb3-
mediated adhesion (IC50=0.014 mM) and had essen-
tially no e€ect on avb3- and avb5-mediated adhesion
(IC50=33±95 mM). Compounds 12 and 14 showed
moderate IC50 values on aIIbb3 (0.13 and 0.2 mM) and
were at least two orders of magnitude less active in
inhibiting avb3- and avb5-mediated adhesion. Com-
pounds 15, 17, 18, and 21 had the highest activity on
avb3- and avb5-mediated adhesion but also inhibited
the most potent (compound 20) being
a mixed
avb3/aIIbb3 inhibitor with an IC50 in the picomolar
range, more active than the highly active peptidic
compound 1.
a
IIbb3-mediated adhesion very e€ectively. Compounds
10, 11, 13, 20 and 22 showed low or no activity on all
three cell lines. Thus, the compounds found to be more
active on isolated integrins were, in general, more active
in whole cell assays. The di€erence in magnitude of
concentration dependency can be ascribed to multi-
valent interactions in the case of the cells.
The nitroaryl ether mimetics showed high activity and
selectivity in the isolated integrin receptor assay and
were, therefore, tested in a cell adhesion assay (Table 2).
The IC50 value for compound 1 as standard was 6.6 mM
for M21 cell adhesion to vitronectin (mediated pre-
dominantly by avb324). Inhibition of UCLA-P3 adhe-
sion to vitronectin (mediated by avb524) resulted in an
IC50 value of 1.7 mM. Inhibition of aIIbb3-mediated
Selected compounds (11, 14 and 16) were tested in the
chick chorioallantoic membrane (CAM) angiogenesis
assay using bFGF as a cytokine.23 The e€ects of these

1192
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
Scheme 4. Synthesis of compounds 11 and 14±19. Reagents and conditions: (a) 1.2 equiv of (COCl)2, PhH, DMF, 0 ꢀC. 6 h, 99%; (b)
1.0 equiv of 29a or 29b, 1.2 equiv of Et3N, CH2Cl2, 0 ꢀC, 2 h, 58 (98%), or 59 (96%); (c) for 60: 2.2 equiv of NaH, 2.2 equiv of 51,
DMF, 25 ꢀC, 8 h, 66%; for 63: 4.0 equiv of NaH, 1.2 equiv of 51, DMF, 25 ꢀC, 4 h, 69% (d) 1.1 equiv of 53, DMF, 25 ꢀC, 4 h, 73%; for
64: 1.9 equiv of 53, NMP, 25 ꢀC, 99%; (e) for 65: 2.2 equiv of NaH, 2.5 equiv of 54, DMF, 25 ꢀC, 12 h, 23%; (f) 1.1 equiv of 52, DMF,
25 ꢀC, 6 h, 83%; for 66: 2.0 equiv of 52, DMF, 25 ꢀC, 20 h, 99%; (g) 50% TFA in CH2Cl2, 25 ꢀC, 30 min, 90±99% for 11, 14±19 after
RP-HPLC. Boc=tert-butoxycarbonyl: TFA=tri¯uoroacetic acid; NMP=N-methyl-2-pyrrolidinone; DMF=dimethylformamide.
compounds, compound 1 and the inactive peptide
cRBADFV (EM-601)4 are shown in Fig. 5. As seen
from these data, compound 11 exhibited excellent inhi-
bition at 30 mg/embryo, while 14 and 16 exhibited mini-
mal inhibition of angiogenesis. Further details regarding
these biological studies are included in the experimental
section.
of small organic molecules based on a nitroaryl ether
sca€old. The synthesis of these compounds relied on a
displacement of a ¯uoride from a nitroaryl system by
nucleophiles carrying oxygen, sulfur or nitrogen atoms,
followed by side-chain construction through amide and
sulfonamide bond formations. The synthesized com-
pounds exhibited varying degrees of inhibition of integ-
rin-mediated cell adhesion with compounds 16 and 19
showing the most potent and selective preference for
Conclusion
a
thermore, compound 11 proved to be an excellent inhi-
bitor or angiogenesis at 30 mg/embryo in the chick
IIbb3- versus avb3- and avb5-mediated adhesion. Fur-
In this article we described the molecular design, che-
mical synthesis and biological investigation of a series

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1193
Scheme 5. Synthesis of compounds 20 and 21. Reagents and conditions: (a) 1.0 equiv of 55, 2.2 equiv of Et3N, DMF, 25 ꢀC, 16 h, 92%;
(b) 50% TFA in CH2Cl2, 25 ꢀC, 4 h, 97%; (c) 1.0 equiv of 56, 2.2 equiv of Et3N, DMF, 12 h, 110% (crude yield); (d) 50% TFA in
CH2Cl2 25 ꢀC, 4 h, 83% after RP-HPLC; TFA=tri¯uoroacetic acid; DMF=dimethylformamide.
Scheme 6. Synthesis of compound 22. Reagents and conditions: (a) NH3, DMF, 25 ꢀC, 5 h, 93%; (b) 10% Pd/C, H2, MeOH, 25 ꢀC,
8 h, 90%; (c) 1.1 equiv of PhNCS, EtOH, 14 h, 69%; (d) 1.0 equiv of HgCl2, 1.0 equiv of Et3N, DMF, 4 h, 81%; (e) 50% TFA in
CH2Cl2, 30 min, 88% yield, after RP-HPLC. TFA=tri¯uroacetic acid; DMF=dimethylformamide.
chorioallantoic membrane (CAM) assay. It is expected
that the use of some of these compounds as delivery
systems may result in the bene®cial localization of
anticancer drugs to the tumor vasculature.24 Further
studies with these compounds is warranted and con-
tinued investigations in the ®eld may prove bene®cial in
biology and medicine.
Experimental
Chemistry general techniques
All reactions were carried out under an argon atmosphere
with dry, freshly distilled solvents under anhydrous
conditions, unless otherwise noted. Tetrahydrofuran
(THF) and diethyl ether (ether) were distilled from

1194
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
Table 1. E€ect of compounds (10±21) on ligand interaction with integrinsa
avb3
avb5
aIIbb3
Compd
IC50 (M)
cQ
IC50 (M)
cQ
IC50 (M)
cQ
GRGDSPK
1
3.2E-07
3.1E-09
1.0E+02
1.0E+00
2.06E-01
>
1.8E-06
>
9.9E+01
1.0E+00
6.0E-06
6.4E-06
9.3E-01
1.0E+00
3.70E-03
2.70E-06
2.00E-05
5.60E-05
1.40E-04
1.10 E-04
2.10E-03
1.40E-04
1.90E-03
3.30E-01
5.60E-06
1.00E-02
7.60E-05
20
18
15
17
21
14
13
16
11
10
19
22
12
8.10E-10
1.30E-09
3.00E-09
3.80E-09
9.5E-09
2.40E-08
1.70E-11
1.30E-10
3.60E-10
9.00E-10
6.7E-10
4.00E-01
9.80E-01
1.3E-06
3.50E-06
2.10E-06
5.00E-06
8.30E-06
>
7.00E-01
1.90E+00
1.20E+00
2.80E+00
4.60E+00
1.20E+00
3.10E+00
1.2E+01
1.40E+01
3.00E+01
3.30E+01
1.30E+02
1.50E+02
4.70E+02
6.00E+02
3.90E-08
4.40E-08
9.30E-08
1.00E-07
4.00E-07
4.80E-07
1.50E-06
1.90E-06
1.40E-08
8.70E-10
1.20E-08
2.10E-06
3.60E-11
6.50E-08
4.90E-10
>
>
9.00E-06
>
>
5.00E+00
>
aBiotinylated ligands vitronectin (avb3 and avb5) or ®brinogen (aIIbb3) were allowed to bind to immobilized integrins in the presence of
the compounds 10±21 (Fig. 3). The concentration necessary for half-maximal inhibition of ligand binding is shown. The peptide
GRGDSPK and compound 1 were included for reference. The data were sorted by IC50 (low to high) on avb3. The `cQ' value desig-
nates the activity of the compound relative to the activity of compound 1. The sign `>', indicates that the IC50 had not been reached
at the maximum concentration tested, (10 mM).
commercial quality and used without further puri®ca-
tion unless otherwise stated.
All reactions were monitored by thin-layer chromato-
graphy carried out on 0.25 mm E. Merck silica gel plates
(60F-254) using UV light as visualizing agent and 7%
ethanolic phosphomolybdic acid or p-anisaldehyde
solution and heat as developing agents. E. Merck silica
gel (60, particle size 0.040±0.063 mm) was used for ¯ash
column chromatography. Preparative thin-layer chro-
matography separations were carried out on 0.25, 0.50
or 1 mm E. Merck silica gel plates (60F-254). Reverse-
phase HPLC was performed on a Waters Model 600E
HPLC instrument utilizing a Vydac 218TP1022 column
with detection at 254 nm using
H2O:CH3CN+0.1% TFA gradient over 40 min.
a
90:10!40:60
NMR spectra were recorded on Bruker DRX-600,
AMX-500, AMX-400 or AC-250 instruments and cali-
brated using residual undeuterated solvent as an inter-
nal reference. The following abbreviations were used to
explain the multiplicities: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; band, several overlapping sig-
nals; b, broad. IR spectra were recorded on a Perkin-
Elmer 1600 series FT-IR spectrometer. High resolution
mass spectra (HRMS) were recorded on a VG ZAB-
ZSE mass spectrometer under fast atom bombardment
(FAB) conditions. Electrospray mass spectra were
recorded on a Perkin Elmer Science API III mass
spectrometer.
Figure 5. E€ect of compounds on bFGF induced CAM
angiogenesis.
sodium-benzophenone,
and
methylene
chloride
(CH2Cl2), benzene (PhH), and toluene from calcium
hydride. Anhydrous solvents were also obtained by
passing them through commercially available activated
alumina columns. Yields refer to chromato-
graphically and spectroscopically (1H NMR) homo-
geneous materials, unless otherwise stated. All solutions
used in workup procedures were saturated unless other-
wise noted. All reagents were purchased at highest

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1195
Table 2. E€ect of compounds (10±21) on cell adhesiona
NMR (150 MHz, CDCl3): d 171.9, 171.2, 155.7, 135.4,
128.5, 128.3, 128.2, 80.1, 67.4, 50.3, 37.4, 28.3; FAB-
HRMS (M+Na+) calcd 345.1426, found 345.1421.
Compd
M21/VNb UCLA-P3/VNb M21-L-IIb/Fgb
IC50 (M)
IC50 (M)
IC50 (M)
Compound 26. To a stirred solution of bis[tri¯uoro-
acetoxy]phenyl iodine (2.0 g, 4.7 mmol) in DMF:H2O
(12 mL:12 mL) compound 25 (1.0 g, 3.1 mmol) was
added at room temperature. After 15 min, pyridine
(0.5 mL, 6.2 mmol) was added and stirring was con-
tinued for 3 h. The solvent was removed under reduced
pressure and the residue dissolved in water (30 mL). The
solution was washed with ether and the aqueous layer
was basi®ed with 1 N NaOH and extracted with
dichloromethane. The solvent was removed under
reduced pressure to give an oily residue. Puri®cation by
¯ash column chromatography (10% MeOH in CH2Cl2)
gave amine 26 as a yellow oil (0.37 g, 41%). Rf=0.11
(2.5% methanol in ethyl acetate); IR (thin ®lm): Vmax
3366, 3313, 3064, 2979, 2934, 1688, 1518, 1501, 1456,
GRGDSPK
1
3.3E-05
6.6E-06
1.3E-06
2.0E-06
3.3E-06
8.7E-06
1.1E-05
1.4E-05
4.0E-05
4.5E-05
6.0E-05
7.5E-05
9.5E-05
>1.0E-04
>1.0E-04
4.0E-05
1.7E-06
8.3E-07
6.0E-06
3.5E-06
1.8E-06
9.0E-06
4.0E-05
1.5E-05
3.3E-05
1.5E-05
4.5E-05
4.7E-05
>1.0E-04
5.0E-05
7.5E-05
>1.0E-04
1.5E-07
5.0E-07
2.0E-08
4.4E-08
1.3E-07
1.0E-05
2.5E-06
1.4E-08
4.5E-05
2.5E-06
1.4E-08
3.5E-05
2.0E-07
15
21
18
17
14
20
13
16
10
11
19
22
12
aCells (25000) were allowed to adhere to immobilized ligands in
the presence of the compounds as described in the text. The
concentration resulting in half-maximal inhibition of cell adhe-
sion (IC50) is shown. The data were sorted by IC50 (low to high)
on avb3 mediated adhesion of M21 cells.
1393, 1368, 1324, 1254, 1204, 1166, 1055, 1002, 838, 800,
1
743, 692 cm
;
1H NMR (500 MHz, CDCl3): d 7.36±
7.28 (m, 5H, Ph), 6.36 (bm, 2H, NH2), 6.06 (d,
J=7.5 Hz, 1H, NHCO2), 5.18 (d, J=12.0 Hz, 1H,
CHHPh), 5.13 (d, J=12.0 Hz, 1H, CHHPh), 4.52±4.43
(bm, 1H, CHCH2), 3.35 (bdd, J=12.5 Hz, 1H,
CHCHH), 3.24 (bdd, J=6.5, 12.5 Hz, 1H, CHCHH),
1.32 (s, 9H, tBu); 13C NMR (125 MHz, CDCl3): d 170.0,
155.9, 134.8, 128.5, 128.4, 128.3, 80.6, 67.7, 52.9, 41.6,
28.0; FAB-HRMS (M+H+) calcd 295.1658, found
295.1650.
bM21 cell line expresses avb3/avb5 (10:1), UCLA-P3 expresses
avb5, M21-L-IIb expresses aIIbb3, VN=vitronectin; Fg=®brino-
gen.
Preparation of amino acid derivatives 26, 29a, and 29b
Compound 25. To a solution of t-butoxycarbonyl-(l)-
asparagine 24 (12.6 g, 50 mol) in MeOH (200 mL) was
added water (20 mL). The solution was neutralized with
a 20% aqueous solution of Cs2CO3 (57 mL) and then
evaporated to dryness. The resulting residue was taken
up in DMF (50 mL) and then azeotropically dried by
evaporation to dryness. The cesium salt was then taken
up in DMF (125 mL) followed by addition of benzyl
bromide (6.5 mL, 55 mmol). The mixture was stirred at
room temperature for 6 h, evaporated to dryness and
the residue triturated with water (500 mL). The solid
was dissolved in ethyl acetate (150 mL) and the organic
phase was washed with water (75 mL), dried over
Na2SO4 and the solvent was removed under reduced
pressure. The crude ester was recrystallized from ethyl
acetate/hexane to give 25 (15.1 g, 88%) as a colorless
Compound 27a. To a solution of (l)-asparagine (23)
(10.00 g, 75.7 mmol) in H2O:dioxane (50 mL:50 mL) was
added NaOH (3.40 g, 85.0 mmol) at 0 ꢀC. After 15 min
at 0 ꢀC, phenylsulfonylchloride (10.6 mL, 84.0 mmol)
was added followed by addition of a solution of NaOH
(3.40 g, 85.0 mmol) in H2O (50 mL) at 0 ꢀC. After
30 min, the cooling bath was removed and the solution
was concentrated to ca. 50 mL under reduced pressure.
The aqueous phase was extracted with ethyl acetate
(2Â50 mL). The aqueous layer was acidi®ed at 0 ꢀC with
concd aqueous HCl (pH&1) while the protected amino
acid precipitated. The resulting solid was collected by
®ltration and washed with H2O (20 mL). Overnight
drying in an oven at ca. 50 ꢀC gave 27a as a colorless
solid (14.6 g, 71%). The crude product was used without
further puri®cation. IR (KBr): Vmax 3495, 3338, 3260,
solid. IR (KBr): Vmax 3401, 3349, 3204, 2982, 2935,
1
1723, 1648, 1578, 1449, 1325, 1261, 1202, 1166,
1
1741, 1688, 1657, 1524, 1293, 1169, 1055 cm
;
1H
1086 cm
;
1H NMR (500 MHz, methanol-d4): d 7.88±
NMR (500 MHz, CDCl3): d 7.36±7.32 (m, 5H, Ph),
5.73 (d, J=8.5 Hz, 1H, NHCO2), 5.59 (bs, 1H,
CONHH), 5.40 (bs, 1H, CONHH), 5.20 (d,
J=12.5 Hz, 1H, CHHPh), 5.17 (d, J=12.5 Hz, 1H,
CHHPh), 4.56 (ddd, J=4.0, 5.0, 8.5 Hz, 1H, CHCH2),
2.95 (dd, J=5.0, 16.5 Hz, 1H, CHCHH), 2.76 (dd,
7.86 (m, 2H, Ph), 7.60±7.57 (m, 1H, Ph), 7.54±7.51 (m,
2H, Ph), 4.23 (t, 1H, J=6.0 Hz, CHCO2H), 2.66 (dd,
1H, J=6.0, 15.5 Hz, CHH(C=O)NH2), 2.60 (dd,
J=6.0, 15.5 Hz, CHH(C=O)NH2); 13C NMR
(125 MHz, methanol-d4): d 174.3, 173.6, 142.1, 133.7,
130.0, 128.2, 53.9, 39.6; FAB-HRMS (M+H+) calcd
273.0545, found 273.0540.
t
J=4.0, 16.5 Hz, 1H, CHCHH), 1.42 (s, 9H, Bu); 13C

1196
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
Compound 27b. Compound 27b was prepared by the
same procedure as for 27a. Crude yield: 16.06 g (66%).
IR (KBr): Vmax 3424, 3289, 2925, 1851, 1713, 1673,
2H, naphthyl), 8.04 (d, J=8.5 Hz, 1H, naphthyl), 7.85
(dd, J=2.0, 8.5 Hz, 1H, naphthyl), 7.70 (ddd, J=1.5,
7.0, 8.0 Hz, 1H, naphthyl), 7.66 (ddd, J=1.0, 7.0,
8.0 Hz, 1H, naphthyl), 7.45 (bm, 1H, NHSO2), 3.54±
3.19 (bm, 2H, NH2), 3.23 (dd, superimposed, J=4.5,
9.5 Hz, 1H, CHCH2), 3.04 (dd, J=4.5, 12.0 Hz, 1H,
CHCHH), 2.83 (dd, J=9.5, 12.0 Hz, 1H, CHCHH); 13C
NMR (125 MHz, DMSO-d6): d 169.3, 136.1, 134.3,
131.6, 129.4, 129.2, 128.9, 128.0, 127.8, 127.6, 122.5,
52.7, 41.6; FAB-HRMS (M+H+) calcd 295.0753,
found 295.0761
1502, 1399, 1333, 1258, 1223, 1191, 1159, 1127, 1075,
1
1023, 964, 866, 822, 794, 714, 669, 638, 548, 477 cm
;
1H NMR (500 MHz, DMSO-d6): d 8.40 (d, J=1.0 Hz,
1H, naphthyl), 8.15 (d, J=9.0 Hz, 1H, NHSO2), 8.12 (d,
J=8.0 Hz, 1H, naphthyl), 8.07 (d, J=9.0 Hz, 1H,
naphthyl), 8.01 (d, J=8.0 Hz, 1H, naphthyl), 7.81 (dd,
J=1.5, 8.8 Hz, 1H, naphthyl), 7.68 (ddd, J=1.5, 6.8,
7.5 Hz, 1H, naphthyl), 7.64 (ddd, J=1.5, 6.8, 7.5 Hz,
1H, naphthyl), 7.33 (bs, 1H, CONHH), 6.87 (bs, 1H,
CONHH), 4.16 (bm, 1H, CHCH2), 2.47 (dd, J=7.0,
15.5 Hz, 1H, CHCHH) 2.28 (dd, J=6.5, 15.5 Hz, 1H,
CHCHH); 13C NMR (125 MHz, DMSO-d6): d 172.0,
170.5, 138.4, 134.1, 131.6, 129.2, 129.0, 128.6, 127.8,
127.4, 127.1, 122.6, 52.5, 38.0; FAB-HRMS (M+H+)
calcd 323.0702, found 323.0708.
Compound 29a. A sealed tube equipped with a magnetic
stirring bar was charged with 28a (4.88 g, 20.0 mmol)
and 75 mL of anhydrous dimethoxyethane. After addi-
tion of 3.0 mL of concd H2SO4 the reaction mixture was
cooled to 78 ꢀC under an atmosphere of argon and
40 mL of isobutylene was condensed into the sealed
tube. After sealing the cooling bath was removed and
the reaction mixture was stirred for 48 h at room tem-
perature. The reaction mixture was poured into 100 mL
of ice water. The aqueous solution was extracted with
diethyl ether (40 mL) and then the pH of the aqueous
phase adjusted to 12±13 with 6 N aqueous NaOH. The
free amine was extracted with ethyl acetate (4Â50 mL)
and the combined organic extracts were washed succes-
sively with saturated aqueous NaHCO3 solution
(50 mL), 5% aqueous KHSO4 solution (50 mL) and
brine (50 mL). The organic phase was dried over
MgSO4, ®ltrated and the solvent removed in vacuo to
give 29a (3.32 g, 55.5%) as an o€-white solid. The crude
product was used without further puri®cation. Rf=0.27
(silica gel, ethyl acetate); IR (KBr): Vmax 3372, 3295,
Compound 28a. To a round-bottom ¯ask equipped with
a magnetic stirring bar was added an aqueous solution
of NaOH (11.15 g, 280.0 mmol) in water (50 mL) and
cooled to 0 ꢀC. Bromine (2.60 mL, 50.0 mmol) was
added dropwise within 5 min and the reaction mixture
was stirred for a further 5 min at this temperature. A
solution of the protected amino acid 27a (10.44 g,
38.0 mmol) in a solution of NaOH (3.10 g, 70.0 mmol,
30 mL H2O) was added in one portion at 0 ꢀC. Stirring
was continued at this temperature for 20 min and upon
removal of the cooling bath the reaction mixture was
heated to 90 ꢀC for an additional 30 min. After cooling
to 0 ꢀC the pH of the reaction mixture was adjusted to 7
with concentrated and 1 M aqueous HCl. The resulting
colourless precipitate was collected by ®ltration. The
residue was washed with water, dried overnight in an
oven at ca. 50 ꢀC to give 28a as a colorless solid (6.95 g,
75%). The crude product was used without further
puri®cation. IR (KBr): Vmax 3509, 3299, 3058, 2936,
2981, 2932, 1726, 1452, 1337, 1261, 1161, 1094, 945, 898,
1
845, 753 cm
;
1H NMR (500 MHz, CDCl3): d 7.86±
7.84 (m, 2H, Ph), 7.58±7.54 (m, 1H, Ph), 7.51±7.48 (m,
1H, Ph), 3.76 (dd, J=4.0, 5.5 Hz, 1H, CHCH2), 3.02
(dd, J=9.0, 13.0 Hz, 1H, CHH), 2.88 (dd, J=5.5,
13.0 Hz, 1H, CHH), 1.27 (s, 9H, tBu); 13C NMR
(125 MHz, CDCl3): d 169.3, 139.7, 132.8, 129.1, 127.2,
82.8, 58.8, 44.9, 27.7; FAB-HRMS (M+H+) calcd
301.1222, found 301.1211.
1636, 1596, 1522, 1446, 1413, 1355, 1340, 1300, 1246,
1
1163, 1094, 1028, 924 cm
;
1H NMR (500 MHz,
DMSO-d6): d 7.84±7.82 (m, 2H, Ph), 7.66±7.63 (m, 1H,
Ph), 7.60±7.56 (m, 2H, Ph), 7.58 (bm, 1H, NHSO2Ph),
3.35 (bs, 2H, NH2), 3.17 (dd, J=4.5, 9.5 Hz, 1H,
CHCH2), 3.01 (dd, J=2.5, 12.0 Hz, 1H, CHH), 2.80
(dd, J=9.5, 12.0 Hz, 1H, CHH); 13C NMR (125 MHz,
DMSO-d6): d 169.4, 139.1, 132.7, 129.2, 126.8, 52.7,
41.7; FAB-HRMS (M+Na+) calcd 245.0596, found
245.0599.
Compound 29b. Compound 29b was prepared by the
same procedure as for 29a. Crude yield: 4.56 g (51%) as
an o€-white solid. Rf=0.25 (silica gel, ethyl acet-
ate+2% Et3N); IR (KBr): Vmax 3360, 3264, 3057, 2981,
2935, 2873, 2747, 1731, 1588, 1501, 1455, 1339, 1254,
1220, 1157, 1076, 952, 926, 823, 782, 747, 663, 619, 552,
1
Compound 28b. Compound 28b was prepared by the
same procedure as for 28a. Crude yield (11.88 g, 81%)
as a beige-coloured solid. IR (KBr): Vmax 3338, 3241,
3056, 2831, 2601, 1651, 1604, 1513, 1463, 1404, 1382,
481 cm 1; H NMR (500 MHz, CDCl3): d 8.40 (bs, 1H,
naphthyl), 7.92 (bd, J=8.5 Hz, 2H, naphthyl), 7.86 (d,
J=8.0 Hz, 1H, naphthyl), 7.82 (dd, J=1.5, 8.5 Hz, 1H,
naphthyl), 7.64±7.55 (2Âddd, superimposed, J=1.0, 7.0,
8.5 Hz, 2H, naphthyl), 3.84 (dd, J=4.2, 5.8 Hz, 1H,
CHCH2), 3.30±2.60 (bm, superimposed, 2H, NH2), 3.03
(dd, J=4.2, 13.4 Hz, 1H, CHCHH), 2.88 (dd, J=5.8,
1335, 1151, 1076, 1039, 985, 955, 931, 913, 855, 814, 787,
1
749, 657, 618, 550, 480 cm
;
1H NMR (500 MHz,
DMSO-d6): d 8.48 (bs, 1H, naphthyl), 8.16±8.10 (2 bd,

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
t
1197
13.4 Hz, 1H, CHCHH), 1.08 (s, 9H, Bu); 13C NMR
(125 MHz, CDCl3): d 169.2, 136.3, 134.9, 132.0, 129.5,
129.2, 128.9, 128.6, 127.8, 127.5, 122.4, 82.9, 58.6, 44.6,
27.5; FAB-HRMS (M+H+) calcd 351.1379, found
351.1371.
was then extracted with CH2Cl2 (3Â70 mL). The com-
bined organic extracts were dried over MgSO4, ®ltered
and the solvent removed under reduced pressure to give
33 as a yellow solid (3.50 g, 99%). Rf=0.10 (silica gel,
60% ethyl acetate in hexanes); IR (KBr): Vmax 3087,
2964, 2877, 2659, 2539, 2120, 1699, 1616, 1534, 1429,
1359, 1282, 1163, 1138, 1079, 1039, 1002, 929, 847, 763,
Preparation of compounds 10±13
1
687, 642, 546 cm 1; H NMR (500 MHz, methanol-d4):
Compound 31. To a suspension of the acid 30 (5.0 g,
27 mmol) in toluene was added trimethylorthoacetate
(17 mL, 135 mmol). The mixture was heated to 80 ꢀC for
12 h and the solvent removed under reduced pressure to
give 31 as a colorless solid (5.26 g, 98%). Rf=0.46 (silica
gel, 25% ethyl acetate in hexane); IR (KBr): Vmax 3061,
d 8.40 (d, J=2.0 Hz, 1H, Ar), 8.22 (dd, J=2.0, 9.0 Hz,
1H, Ar), 7.37 (d, J=9.0 Hz, 1H, Ar), 4.37 (t, J=4.5 Hz,
2H, OCH2), 3.67 (t, J=5.0 Hz, 2H, CH2N3); 13C NMR
(125 MHz, methanol-d4): d 167.4, 156.0, 140.9, 136.4,
127.9, 124.9, 115.7, 70.3, 51.1; FAB-HRMS (M+Na+)
calcd 275.0392, found 275.0395.
2960, 1714, 1614, 1542, 1438, 1351, 1297, 1262, 1233,
1
1130, 871, 755 cm
;
1H NMR (500 MHz, CDCl3): d
Compound 34. To a solution of amine 26 (0.33 g,
1.10 mmol) and acid 33 (0.286 g, 1.10 mmol) in CH2Cl2
(30 mL) was added a catalytic amount of 4-DMAP
(0.03 g, 0.22 mol) and DCC (0.26 g, 1.1 mol) at room
temperature. The reaction mixture was stirred for 4 h at
this temperature and the precipated dicyclohexyl urea
was then ®ltered and the ®ltrate washed successively
with water, saturated aqueous NaHCO3-solution and
brine. The organic solvent was removed under reduced
pressure to give an oil which after puri®cation by ¯ash
column chromatography (silica gel, 60% ethyl acetate in
hexanes) gave amide 34 as a yellow solid (2.48 g, 82%).
Rf=0.28 (silica gel, 60% ethyl acetate in hexanes); IR
(KBr): Vmax 3343, 2977, 2933, 2112, 1738, 1710, 1619,
1531, 1498, 1366, 1333, 1280, 1161, 1084, 1047 cm 1; 1H
NMR (500 MHz, CDCl3): d 8.23 (d, J=2.0 Hz, 1H, Ar),
7.98 (dd, J=2.0, 11.0 Hz, 1H, Ar), 7.40 (bt, 1H,
NHCO), 7.39±7.31 (m, 5H, Ph), 7.09 (d, J=11.0 Hz,
1H, Ar), 5.67 (d, J=8.0 Hz, 1H, NHCO2), 5.21 (s, 2H,
CH2Ph), 4.60±4.50 (bm, 1H, CHCH2), 4.30 (t,
J=6.0 Hz, 2H, OCH2), 3.95±3.85 (bm, 1H, CHCHH),
3.78±3.70 (bm, superimposed, 1H, CHCHH), 3.70 (t,
4
4
8.74 (dd, J (1H-1H)=2.0 Hz, J (1H-19F)=7.0 Hz, 1H,
Ar), 8.32 (ddd, 4J(1H-1H)=2.0 Hz, 4J (1H-19F)= 4.5 Hz,
3J(1H-1H)=9.0Hz, 1H, Ar), 7.39 (dd, 3J(1H-1H)=9.0 Hz,
3J(1H-19F)=10.5 Hz, 1H, Ar); 13C NMR (125 MHz,
CDCl3): d 164.1, 159.1, 156.9, 136.5, 127.8, 118.8, 118.7,
52.9; FAB-HRMS (M+H+) calcd 200.0281, found
200.0286.
Compound 32. To a solution of 31 (4.0 g, 20 mmol) in
DMF (10 mL) were added ca. 4 g of 4 A molecular sieves
and 4.46 g (0.22 mmol) of N3(CH2)2OTBS at room tem-
perature. After 10 min, 2 mL (2.0 mmol) of a 1 M solu-
tion of TBAF in THF was added. The mixture was
stirred for 4 h at room temperature and then ®ltered
through a short path of celite1. The ®ltrate was taken
up in ethyl acetate (100 mL) and successively washed
with water, saturated aqueous NaHCO3-solution and
brine. The organic extract was dried over MgSO4, ®l-
tered and the solvent removed under reduced pressure
to give a yellow oil. Flash column chromatography
(silica gel, 40% ethyl acetate in hexanes) gave 32 as a
yellow solid (3.85 g, 73%). Rf=0.35 (silica gel, 40%
ethyl acetate in hexanes); IR (KBr): Vmax 2950, 2938,
2114, 1713, 1620, 1536, 1438, 1349, 1303, 1276, 1247,
1161, 1136, 918, 760 cm 1; 1H NMR (500 MHz, CDCl3):
d 8.54 (d, J=2.0 Hz, 1H, Ar), 8.22 (dd, J=2.0, 9.0 Hz,
1H, Ar), 7.13 (d, J=9.0 Hz, 1H, Ar), 4.32 (t, J=5.0 Hz,
2H, OCH2), 3.94 (s, 3H, OCH3), 3.71 (t, J=5.0 Hz, 2H,
CH2N3); 13C NMR (125 MHz, CDCl3): d 164.7, 154.7,
135.3, 127.3, 123.3, 114.0, 68.8, 52.5, 49.7; FAB-HRMS
(M+Na+) calcd 289.0549, found 289.0553.
t
J=6.0 Hz, 2H, CH2N3), 1.43 (s, 9H, Bu); 13C NMR
(125 MHz, CDCl3): d 170.0, 164.9, 153.8, 139.8, 135.0,
133.0, 128.7, 128.6, 126.9, 124.6, 114.3, 81.0, 68.8, 67.9,
49.8, 33.8, 28.2, 25.5, 24.8; FAB-HRMS (M+Cs+)
calcd 661.1023, found 661.1050.
Compounds 35ab. To a solution of the azide 34 (50 mg,
0.095 mmol) in a mixture of THF:H2O (8 mL THF:
0.04 mL H2O) was added triphenylphosphine (50 mg,
0.19 mmol). The reaction mixture was stirred at room
temperature for 14 h and the solvent was removed under
reduced pressure. Puri®cation of the crude residue by
¯ash column chromatography (silica gel, 20% methanol
in dichloromethane) gave two major fractions (total
yield 80%). Fraction 1: 17 mg (yellowish oil, 40%);
fraction 2: 17 mg (yellowish oil, 40%). Fraction 1 was
positive in ninhydrin test while fraction 2 was negative.
Fraction 1 35a Rf=0.48 (silica gel, 20% methanol in
Compound 33. To a solution of 32 (3.85 g, 14.0 mmol) in
.
1,4-dioxane:water (90 mL:30 mL) was added LiOH H2O
(1.2 g, 28 mmol). The reaction mixture was stirred at
room temperature for 4 h and then 40 mL of a saturated
aqueous NH4Cl-solution was added. The organic sol-
vent was removed under reduced pressure to give a yel-
low slush. The slush was taken up in water and acidi®ed
with aqueous 1 M KHSO4-solution. The aqueous layer
1
dichloromethane); H NMR (500 MHz, CDCl3): d 8.22

1198
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
(m, 1H, Ar), 7.94 (d, J=9.0 Hz, 1H, Ar), 7.43±7.30 (m,
6H), 7.07 (d, J=9.0 Hz, 1H, Ar), 5.80 (d, J=6.0 Hz, 1H,
NHBoc), 5.19 (s, 2H, CH2Ph), 4.54 (bm, 1H,
CHNHBoc), 4.21 (bm, 2H, CH2OAr), 3.87±3.76 (m,
2H), 3.19 (s, 2H, CH2NH2), 2.75 (bs, 2H, NH2), 1.41 (s,
9H, tBu); FAB-HRMS (M+H+) calcd 503.2142,
found 503.2162. Fraction 2 35b: Rf=0.86 (silica gel,
20% methanol in dichloromethane); 1H NMR
(500 MHz, CDCl3): d 8.46 (m, 1H, Ar), 8.42 (t,
J=6.5 Hz, 1H, NHAr), 7.79 (dd, J=2.5, 11.0 Hz, 1H,
Ar), 7.38±7.28 (m, 5H, Ar), 7.21 (m, 1H, NHCO), 6.80
(d, J=11.0 Hz, 1H, Ar), 5.85 (d, J=9.0 Hz, 1H, NHBoc),
5.19 (s, 2H, CH2Ph), 4.53 (m, 1H, CHNHBoc), 3.92
(t, J=6.5 Hz, 2H, CH2OH), 3.88±3.73 (m, 2H,
CH2NH(CO)), 3.52±3.38 (m, 2H, CH2NHAr), 1.41 (s,
Compound 38a. To a solution of 37 (0.13 g, 0.30 mmol)
in CH2Cl2 (10 mL) was added N,N-diisopropylethyl-
amine (0.07 mL, 0.39mmol) and benzenesulfonyl chloride
(0.034 mL, 0.33 mmol) at room temperature. After 4 h,
the reaction mixture was diluted with CH2Cl2 (10 mL)
and water (10 mL). The layers were seperated and the
organic layer washed with a saturated solution of
sodium bicarbonate and brine and dried (MgSO4). The
solvent was removed in vacuo to give an oil, which after
preparative thin-layer chromatography (silica, 60%
ether in hexanes) gave 38a as an oil (0.13 g, 78%).
Rf=0.43 (silica, 60% ether in hexane); 1H NMR
(500 MHz, CDCl3): d 8.26 (d, J=2.0 Hz, 1H, Ar), 7.98
(dd, J=2.0, 9.0 Hz, 1H, Ar), 7.81 (d, J=8.0 Hz, 2H,
Ar), 7.53 (t, J=8.0 Hz, 1H, Ph), 7.42 (t, J=8.0 Hz, 2H,
Ph), 7.31±7.30 (m, 3H, Ar), 7.22±7.21 (m, 2H, Ar), 7.08
(d, J=9.0 Hz, 1H, Ar), 7.03 (t, J=5.5 Hz, 1H), 5.03 (d,
J=12.0 Hz, 1H, PhCHH), 4.99 (d, J=12.0 Hz, 1H,
PhCHH), 4.29 (t, J=4.5 Hz, 2H, CH2O), 4.16 (dd,
J=4.0, 7.5 Hz, 1H, CHCHH), 3.92±3.87 (m, 1H,
CHCH2), 3.72±3.67 (m, superimposed, 3 H , CHCHH,
CH2N3); 13C NMR (125 MHz, CDCl3): d 169.2, 165.2,
153.7, 139.3, 138.7, 134.3, 133.1, 132.9, 129.1, 128.4,
128.3, 126.9, 126.5, 124.9, 114.1, 68.7, 68.1, 55.4, 49.7,
42.4; FAB-HRMS (M+Cs+) calcd 701.0431, found
701.0442.
t
9H, Bu); FAB-HRMS (M+Cs+) calcd 635.118, found
635.110.
Compound 10. To
0.046 mmol) in a mixture of THF:H2O (6 mL:2 mL) was
a
solution of 35a (0.023 g,
.
added LiOH H2O (4 mg, 0.092 mmol) at room tempera-
ture. The mixture was stirred for 4 h and then acidi®ed
with acetic acid. The solvent was removed under
reduced pressure and the residue used in the next step
without further puri®cation. To a solution of the crude
acid in 2 mL of anhydrous DMF was added N,N-diiso-
propylethylamine (9 mL, 0.05 mmol) and 1H-pyrazole
.
carboxamidine HCl (8 mg, 0.05 mmol). After 16 h, the
Compound 38b. Compound 38b was prepared by the
same procedure as for 38a. Yield (0.031 g, 57%) as an
oil. Rf=0.18 (5% methanol in dichloromethane); IR
solvent was removed under reduced pressure and the
residue was puri®ed by RP-HPLC (C-18) to give 10
(3.25 mg, 13%) as a yellowish solid. tR=20.5 min; 1H
NMR (500 MHz, D2O): d 8.33 (d, 1H, J=2.0 Hz, Ar),
7.98 (dd, 1H, J=2.0, 9.0 Hz, Ar), 7.30 (d, J=9.0 Hz,
1H, Ar), 4.42 (m, 1H, CHNHBoc), 4.34 (t, J=4.0 Hz,
2H, CH2NH(CO)), 3.82 (m, 2H, NH2 (C=NH)
NHCH2), 3.63 (t, 2H, J=4.0 Hz, 2H, CH2O); FAB-
HRMS (M+Cs+) calcd 587.0866, found 587.0895.
(thin ®lm): Vmax 3277, 2930, 2112, 1740, 1652, 1618,
1
1523, 1496, 1348, 1280, 1162, 1125, 984, 910, 772 cm
.
1H NMR (500 MHz, CDCl3): d 8.60 (d, J=11.0 Hz, 1H,
naphthyl), 8.21 (dd, J=2.0, 9.3 Hz, 1H, naphthyl), 8.03
(d, J=2.9 Hz, 1H, Ar), 8.01 (d, J=10.4 Hz, 1H, naph-
thyl), 7.87 (d, J=9.3 Hz, 1H, naphthyl), 7.81 (dd,
J=2.9, 11.0 Hz, 1H, Ar), 7.62 (ddd, J=1.7, 8.7, 8.7 Hz,
1H, naphthyl), 7.54 (ddd, J=1.3, 8.8, 8.8 Hz, 1H,
naphthyl), 7.47 (dd, J=9.4, 10.1 Hz, 1H, naphthyl),
7.28±7.22 (m, 3H, Ph), 7.12±7.07 (m, 2H, Ph), 6.98 (d,
J=11.0 Hz, 1H, Ar), 6.69 (dd, J=7.5 Hz, 1H, NHCO),
6.22 (d, J=9.5 Hz, 1H, NHSO2), 4.89 (d, J=15.0 Hz,
1H, CHHPh), 4.83 (d, J=15.0 Hz, 1H, CHHPh), 4.25
(t, J=6.0 Hz, 2H, OCH2), 4.13 (ddd, J=5.4, 9.4, 9.5 Hz,
1H, CHCH2), 3.74 (ddd, J=5.4, 7.4, 17.5 Hz, 1H,
CHCHH), 3.69 (t, J=6.1 Hz, 2H, CH2N3), 3.67 (ddd,
superimposed, J=7.5, 9.0, 17.5 Hz, 1H, CHCHH); 13C
NMR (125 MHz, CDCl3): d 169.3, 165.1, 153.7, 139.1,
134.8, 134.4, 134.0, 133.5, 132.9, 129.9, 129.0, 128.6,
128.5, 128.3, 127.7, 126.3, 124.8, 124.1, 114.0, 68.7, 67.9,
55.7, 49.8, 42.2; FAB-HRMS(M+Cs+) calcd 751.0587,
found 751.0599.
Compound 37. To a solution of 34 (0.10 g, 0.019 mmol)
in CH2Cl2 (4 mL) at room temperature was added tri-
¯uoroacetic acid (4 mL). The mixture was stirred for 2 h.
The solvent was removed in vacuo to give a yellowish
oil, which after ¯ash chromatography (silica, 5%
methanol in dichloromethane) gave 37 as an oil (0.07 g,
84%). Rf=0.19 (silica, 5% methanol in dichloro-
methane); 1H NMR (500 MHz, CDCl3): d 8.21 (d,
J=2.0 Hz, 1H, Ar), 7.95 (dd, J=2.0, 11.0 Hz, 1H, Ar),
7.39±7.29 (m, 5H, Ar), 7.21 (bm, 1H), 7.05 (d,
J=9.0 Hz, 1H), 5.16 (s, 2H, CH2Ph), 4.27 (t, J=5.0 Hz,
2H, CH2OAr), 3.67 (t, J=5.0 Hz, 2H, CH2N3),
3.95±3.78 (bm, 1H, CHNH2), 3.65±3.52 (bm, 1H,
CHCHH), 4.32±4.31 (bm, 1H, CHCHH); 13C (125 MHz,
CDCl3): d 164.9, 153.7, 139.2, 135.1, 133.1, 128.6,
128.5, 128.4, 127.0, 124.6, 114.2, 68.7, 67.4, 49.7, 33.8,
25.5; FAB-HRMS (M+Cs+) calcd 561.0499, found
561.0507.
Compounds 39a and 41a. Compounds 39a and 41 were
prepared by the same procedure as for 35ab. Yield:
(F1=0.029 g, F2=0.028 g), total yield (80%). F2 was

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1199
positive in ninhydrin test while F1 was not. Rf (F1)
41a=0.39 (silica, 10% methanol in dichloromethane);
1H NMR (500 MHz, CDCl3): d 8.35 (bs, 1H, Ar), 8.31
(bs, 1H, CH2NHAr), 7.76±7.60 (m, superimposed, 3H,
Ar), 7.50 (bs, 1H), 7.38 (t, J=9.0 Hz, 1H, Ar), 7.28 (t,
J=9.0 Hz, 2H, Ar), 7.26±7.13 (m, 5H, Ar), 6.86 (d,
J=11.0 Hz, 1H, Ar), 6.64 (d, J=11.0 Hz, 1H, NHSO2),
4.92 (d, J=15.0 Hz, 1H, PhCHH), 4.86 (d, J=15.0 Hz,
1H, PhCHH), 4.26±4.23 (m, 1H, CHCH2), 3.80±3.67
(m, superimposed, 4H, HOCH2, CHCH2), 3.34±3.31
(bm, CH2NHAr), 2.92 (bs, CH2OH) ; Rf (F2) 39a=0.16
(silica, 20% methanol in dichloromethane); 1H NMR
(500 MHz, CDCl3): d 8.24 (bs, 1H, Ar), 7.94 (d,
J=9.0 Hz, 1H, Ar), 7.80 (d, J=8.0 Hz, 2H), 7.49 (t,
J=8.0 Hz, 1H, Ph), 7.40 (t, J=8.0 Hz, 2H, Ph), 7.30±
7.28 (m, 3H, Ar), 7.19±7.18 (m, superimposed, 3H, Ar,
NH(CO)), 7.02 (d, J=9.0 Hz, 1H, Ar), 5.01 (d,
J=12.5 Hz, 1H, PhCHH), 4.97 (d, J=12.5 Hz, 1H,
PhCHH), 4.18±4.15 (m, superimposed, 3H, CHCH2,
OCH2), 3.86±3.84 (m, 1H, CHCHH), 3.70±3.68 (m, 1H,
CHCHH), 3.15±3.13 (bm, 2H, OCH2NH2), 2.82 (bm,
2H, NH2); 13C NMR (125 MHz, CDCl3): d 169.5, 165.4,
154.4, 139.9, 138.8, 134.4, 133.0, 132.8, 129.0, 138.5,
128.4, 127.0, 126.3, 125.0, 114.1, 71.4, 68.0, 55.4, 42.3,
40.7; FAB-HRMS (M+Cs+) calcd 675.0526, found
675.0546.
125.5, 122.2, 112.5, 70.4, 42.6, 28.7, 23.2. Electrospray
mass spectrum (M+H+) calcd 495, found 495.
Compound 39b. To a solution of the azide 38b (0.031 g,
0.05 mmol) in THF:H2O (8 mL:0.04 mL) was added tri-
phenylphosphine (0.026 g, 0.1 mmol). After stirring at
room temperature for 12 h, the solvent was removed in
vacuo to give a white solid. The solid was purifed by
preparative thin-layer chromatography (silica, 20%
methanol in dichloromethane) to give 39b as an oil
(0.013 g, 44%). Rf=0.1 (silica gel, 10% methanol in
dichloromethane); IR (thin ®lm): Vmax 3361, 3282, 3070,
2922, 2851, 1742, 1650, 1620, 1527, 1456, 1349, 1322,
1
1280, 1162, 1126, 989, 910 cm
;
1H NMR (500 MHz,
CDCl3): d 8.63 (d, J=10.8 Hz, 1H, naphthyl), 8.17 (dd,
J=1.0, 9.1 Hz, 1H, naphthyl), 8.02 (d, J=2.5 Hz, 1H,
Ar), 7.92 (d, J=10.3 Hz, 1H, naphthyl), 7.77 (d,
J=10.3 Hz, 1H, naphthyl), 7.72 (dd, J=2.6, 11.0 Hz,
1H, Ar), 7.58 (dd, J=8.3, 8.3 Hz, 1H, naphthyl), 7.49
(dd, J=7.3, 7.3 Hz, 1H, naphthyl), 7.41 (dd, J=7.5,
7.5 Hz, 1H, naphthyl), 7.23±7.16 (m, 3H, Ph), 7.09±7.04
(m, 2H, Ph), 6.84 (d, J=11.1 Hz, 1H, Ar), 4.83 (d,
J=15.2 Hz, 1H, CHHPh), 4.76 (d, J=15.2 Hz, 1H,
CHHPh), 4.23 (dd, J=5.8, 9.1 Hz, 1H, CHCH2), 4.10±
4.04 (bm, 2H, OCH2), 3.95±3.61 (bm, 5H, CH2NH2,
CHCHH), 3.18±3.05 (bm, 1H, CHCHH); 13C NMR
(125 MHz, CDCl3): d 169.6, 165.3, 154.2, 146.9, 134.6,
134.5, 134.0, 133.9, 133.2, 129.8, 129.0, 128.6, 128.5,
128.4, 128.2, 127.8, 127.0, 125.9, 124.9, 124.2, 124.1,
114.3, 67.6, 55.9, 42.0, 40.5, 29.6; FAB-HRMS
(M+Cs+) calcd 725.0682, found 725.0695.
Compound 11. Compound 11 was prepared by the same
procedure as for 10. Yield: (3.31 mg, 13%). 1H NMR
(500 MHz, methanol-d4): d 8.26 (d, J=2.0 Hz, 1H, Ar),
8.04 (dd, J=2.0, 8.5 Hz, 1H, Ar), 7.82±7.80 (m, 2H, Ph),
7.47±7.36 (m, 4H, Ar, Ph), 4.35 (t, J=5.0 Hz, 2H,
OCH2), 4.19 (dd, J=4.0, 14.0 Hz, 1H, CHCH2), 3.75
(dd, J=4.0, 14.0 Hz, 1H, CHCHH), 3.68 (t, J=5.0 Hz,
2H, CH2NH(C=N)), 3.47 (dd, J=11.0, 14.0 Hz, 1H,
CHCHH).
Compound 13. Compound 13 was prepared by the same
procedure as for 10. Yield: (1.8 mg, 15%) as a yellowish
1
solid. tR=21.2 min; H NMR (500 MHz, methanol-d4):
d 8.63 (d, J=9.0 Hz, 1H, naphthyl), 8.17 (dd, J=1.5,
7.5 Hz, 1H, naphthyl), 7.94 (d, J=8.5 Hz, 1H, naphthyl),
7.88 (d, J=2.5 Hz, 1H, Ar),7.76±7.70 (m, superimposed,
2H, Ar, naphthyl), 7.57 (ddd, J=1.0, 6.5, 9.3 Hz, 1H,
naphthyl), 7.47 (dd, J=7.5, 8.0 Hz, 1H, naphthyl), 7.42
(ddd, J=1.0, 7.0, 7.5 Hz, 1H, naphthyl), 7.25 (d,
J=9.0 Hz, 1H, Ar), 4.36 (t, J=5.0 Hz, 1H, CH2O), 4.18
(dd, J=4.5, 9.5 Hz, 1H, CHCHH), 3.72 (t, J=5.0 Hz,
2H, CH2NH), 3.65 (dd, J=4.5, 13.5 Hz, 1H, CHCHH),
3.41 (dd, J=9.5, 13.5 Hz, 1H, CHCHH); Electrospray
mass spectrum calcd (M+H+) 545, found 545.
Compound 12. To a solution of the benzyl ester 41a
(0.10 g, 0.22 mmol) in THF:H2O (3 mL:1 mL) was added
.
LiOH H2O (18.5 mg, 0.44 mmol) at room temperature.
After stirring for 4 h, the reaction mixture was acidi®ed
with acetic acid and the solvent removed in vacuo to
give the crude acid 42a. To a solution of the acid 42a in
DMF (5 mL) was added N,N-diisopropylethylamine
(38 mL, 0.22 mmol). After stirring at 50 ꢀC for 16 h, the
solvent was removed in vacuo to give an oil, which after
RP-HPLC (C-18) gave 12 (5.4 mg, 5%) as a yellowish
solid. tR=14.9 min; 1H NMR (600 MHz, CDCl3): d 8.26
(d, J=2.0 Hz, 1H, Ar), 7.67±7.65 (m, 2H, Ph), 7.64 (dd,
J=2.0, 9.0 Hz, Ar), 7.24±7.18 (m, 3H, Ph), 7.06 (d,
J=9.0 Hz, Ar), 4.49 (t, J=4.0 Hz, 2H, CH2OH), 4.15
(dd, J=6.0, 10.0 Hz, 1H, CHCH2), 3.86 (t, J=4.0 Hz,
2H, CH2N(C=NH)NH2), 3.70 (dd, J=6.0, 14.0 Hz,
1H, CHCHH), 3.35 (dd, J=10.0, 14.0 Hz, 1H,
CHCHH); 13C NMR (150 MHz, CDCl3): d 167.6, 164.9,
147.2, 141.7, 132.4, 130.5, 129.6, 127.7, 126.8, 125.6,
Preparation of compounds 51±56
Compound 51. To a solution of aminoethanol (43)
(1.0 mL, 16.0 mmol) in DMF (30 mL) was added 1,3-bis-
(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (48)
(4.81 g, 16.0 mmol), triethylamine (4.63 mL, 32.0 mmol)
and mercury(II) dichloride (4.48 g, 16.0 mmol) at room
temperature. After 4 h, the reaction mixture was diluted
with ethyl acetate and ®ltered over a short path of

1200
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
celite1. The ®ltrate was succesively washed with water
(2Â20 mL), brine (20 mL) and dried over MgSO4. After
®ltration and evaporation of the solvent under reduced
pressure the crude compound was puri®ed by ¯ash col-
umn chromatography to give 51 as a colourless solid
(4.95 g, 98%). Rf=0.43 (silica gel, 50% ethyl acetate in
hexanes); IR (KBr): Vmax 3329, 3142, 2977, 2934, 2870,
Compound 54. To a solution of ethylenediamine (54)
(3.45 mL, 51.6 mmol) in DMF (50 mL) was added 1,3-
bis-(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea
(48) (3.00 g, 10.33 mmol), triethylamine (2.88 mL,
20.7 mmol) and mercury(II)chloride (2.81 g, 10.3 mmol)
at room temperature. After 4 h, the reaction mixture
was diluted with 20 mL of ethyl acetate and ®ltered over
a short path of celite1. The ®ltrate was succesively
washed with H2O (2Â50 mL), brine (50 mL) and dried
over MgSO4. Flash column chromatography (silica gel,
20% MeOH in ethyl acetate+2% Et3N) gave 54 as a
colourless solid (1.60 g, 51.2%). Rf=0.30 (silica gel,
20% MeOH in ethyl acetate+2% Et3N); IR (KBr):
Vmax 3446, 3389, 3259, 2978, 2819, 1728, 1706, 1656,
1724, 1644, 1443, 1412, 1360, 1299, 1103, 1052, 1027,
1
864, 809, 778 cm
;
1H NMR (500 MHz, CDCl3): d
11.48 (bs, 1H, NHCO2), 8.66 (m, 1H, CH2NH), 4.54
(bs, 1H, OH), 3.74 (t, J=4.5 Hz, 2H, CH2OH), 3.54 (dt,
t
J=5.5, 5.5 Hz, 2H, CH2NH), 1.47 (s, 9H, Bu), 1.45 (s,
t
9H, Bu); 13C NMR (125 MHz, CDCl3): d 162.8, 157.4,
153.1, 83.5, 79.2, 63.1, 44.4, 28.2, 28.0; FAB-HRMS
(M+H+) calcd 304.1872, found 304.1878.
1626, 1521, 1485, 1365, 1253, 1171, 1093, 1049, 888, 802,
;
1H NMR (500 MHz, CDCl3): d
1
738, 699, 562 cm
Compound 52. To a solution of piperazine (44) (3.45 mL,
12.0 mmol) in DMF (10 mL) was added 1,3-bis-(tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea (48) (0.87 g,
3.00 mmol) at room temperature. After 14 h, the reaction
mixture was diluted with ethyl acetate and water. The
layers were separated and the organic layer was washed
succesively with water (2Â20 mL), brine (20 mL) and
dried over Na2SO4. The solvent was removed under
reduced pressure to give 52 as a colorless solid (0.93 g,
95%). Rf=0.34 (silica gel, 10% methanol in dichloro-
methane); IR (KBr): Vmax 3294, 2980, 2931, 2856, 1749,
11.38 (bs, 1H, (C=N)NH(C=O)), 8.61 (bt, 1H,
CH2NH(C=N)), 3.45 (bdt, J=5.5, 5.5 Hz, 2H,
CH2NH), 2.85 (t, J=5.5 Hz, 2H, CH2NH2), 1.46 (s, 9H,
tBu) ; 13C NMR (125 MHz, CDCl3): d 163.4, 156.4,
153.1, 83.1, 79.2, 41.8, 40.9, 28.2; FAB-HRMS
(M+H+) calcd 303.2032, found 303.2037.
Compound 55. To a solution of phenylenediamine
(1.08 g, 0.01 mol) in 5.5 N HCl (10 mL) was added b-
alanine (47) (1.125 g, 0.015 mol) at room temperature.
The reaction mixture was re¯uxed for 24 h and then
allowed to cool to room temperature. The solvent was
removed in vacuo to give a precipitate, which was ®l-
tered and washed with ether (1.70 g, 73%). Rf=0.12
(20% methanol in dichloromethane); 1H NMR
(500 MHz, D2O): d 7.73±7.72 (m, 2H, Ar), 7.55±7.53 (m,
2H, Ar), 3.61±3.55 (m, 4H, CH2CH2); FAB-HRMS
(M+H+) calcd 162.1031, found 162.1029.
1664, 1605, 1527, 1448, 1367, 1305, 1230, 1149, 1116,
1
1019, 893, 842, 730, 682 cm
.
1H NMR (500 MHz,
methanol-d4): d 3.48 (t, J=5.0 Hz, 4H, (CH2)2N(C=N),
t
2.83 (t, J=5.0 Hz, 4H, (CH2)2NH), 1.46 (s, 9H, Bu);
13C NMR (125 MHz, methanol-d4): d 154.4, 81.4, 48.2,
46.0, 28.6; FAB-HRMS (M+Na+) calcd 341.1226,
found 341.1235.
Compound 53. To a solution of aminoethanethiol (45)
(114 mg, 1.00 mmol) in DMF (5 mL) was added N,N0-
bis-tert-butoxycarbonylthiourea (49) (276 mg, 1.00 mmol)
and triethylamine (2.79 mL, 2.0 mmol) at room tem-
perature. After 14 h, the reaction mixture was diluted
with 5 mL of H2O and extracted with ethyl acetate
(3Â10 mL). The combined organic extracts were washed
with brine and dried over MgSO4. After ®ltration and
evaporation of the solvent under reduced pressure the
crude compound was puri®ed by ¯ash column chroma-
tography (silica gel, 25% diethyl ether in hexanes) to
give 53 as an airsensitive colorless solid (191 mg, 59.8%).
Rf=0.16 (silica, 25% diethyl ether in hexanes); IR (KBr):
Vmax 3327, 3132, 2978, 2931, 1726, 1643, 1565, 1431,
1363, 1329, 1280, 1227, 1133, 1088, 1058, 855, 809, 760,
606 cm 1; 1H NMR (500 MHz, CDCl3): d 11.45 (bs, 1H,
(C=N)NH(C=O)), 8.61 (bt, J=5.9 Hz, 1H, CH2NH),
3.74 (bdt, J=6.0, 6.5Hz, 2H, CH2NH), 2.85 (t, J=6.5 Hz,
Compound 56. To a solution of ethylenediamine (46)
(1.0 mL, 0.015 mol) in DMF (10 mL) was added 2-(3,5-
dimethylpyrazolyl)-4,5-dehydroimidazole hydrobromide
(50) (3.67 g, 0.015 mol) and N,N-diisopropylethylamine
(2.61 mL, 0.015 mol) at room temperature. After stirring
for 11 h, ether (12 mL) was added to the reaction mix-
ture and a white precipitate formed. The precipitate was
®ltered and washed with ether to give 56 (1.59 g, 51%).
IR (KBr): Vmax 3164, 1681, 1599, 1484, 1287, 1211,
1137, 1069, 952 cm 1; 1H NMR (500 MHz, D2O): d 3.54
(bs, 4H, NHCH2CH2NH), 3.17 (t, J=6.0 Hz, CH2NH),
2.65 (t, J=6.0 Hz, CH2NH2); 13C NMR (125 MHz,
D2O): d 160.9, 45.3, 43.6, 40.3; FAB-HRMS (M+H+)
calcd 129.1140, found 129.1134.
Preparation of compounds 11 and 14±19
Compound 58. To a solution of 3-nitro-4-¯uoro benzoic
acid (30) (1.59 g, 8.57 mmol) in benzene (40 mL) was
added DMF (0.03 mL, 0.40 mmol) and oxalylchloride
(3.73 mL, 20.2 mmol) at 0 ꢀC. After 6 h, the solvent was
t
2H, CH2SH), 1.47 (s, 9H, Bu); 13C NMR (125 MHz,
CDCl3): d 163.4, 156.1, 153.1, 83.2, 79.3, 39.2, 37.0,
28.3, 28.1; FAB-HRMS calcd only S-S-dimer.

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1201
removed in vacuo. The resulting yellow viscous oil
(1.73 g, 8.57 mmol) was dissolved in CH2Cl2 (20 mL).
The solution was cooled to 0 ꢀC and triethylamine
(1.28 mL, 9.20 mmol) was added. A solution of the pro-
tected 2-amino alanine tert-butylester 29a (2.32 g,
7.70 mmol) in CH2Cl2 (40 mL) was added. After 4 h, the
reaction mixture was diluted with water and the aque-
ous phase was extracted with dichloromethane
(2Â50 mL) after phase separation. The combined
organic extracts were washed with saturated aqueous
NaHCO3-solution and dried over MgSO4. After ®ltra-
tion and evaporation of the solvent under reduced
pressure the crude compound was puri®ed by ¯ash col-
umn chromatography (silica gel, 45% ethyl acetate in
hexanes) to give 58 as a yellow foam (3.90 g, 98%).
Rf=0.19 (silica gel, 40% ethyl acetate in hexanes); IR
FAB-HRMS (M+Cs+) calcd 650.0373, found
650.0358.
Compound 60. To a round bottom ¯ask equipped with a
magnetic stirring bar was placed NaH (60% suspension
in mineral oil) (0.16 g, 3.96 mmol) and THF (10 mL) at
0 ꢀC . To the stirred suspension was added a solution of
51 (0.55 g, 1.80 mmol) in THF (5 mL). Stirring was con-
tinued at this temperature for an additional 30 min and
the resulting grey suspension was ready for use. A round
bottom ¯ask equipped with a magnetic stirring bar was
charged with the aromatic ¯uoride 58 (0.1 g, 0.30 mmol)
and DMF (10 mL). The solution was cooled to 0 ꢀC and
5.5 mL of the previously prepared suspension was added
by means of a syringe. After 8 h at 0 ꢀC the reaction was
stopped by addition of water (10 mL) and diluted with
ethyl acetate. The aqueous phase was separated and
extracted with ethyl acetate (3Â25 mL) The combined
organic extracts were washed successively with H2O
(2Â10 mL) and brine (10 mL) and dried over MgSO4.
After ®ltration and evaporation under reduced pressure
the residue was puri®ed by ¯ash column chromatog-
raphy (silica, 60% ethyl acetate in hexanes) to give 60 as
a yellowish foam (0.15 g, 66%). Rf=0.18 (silica gel, 50%
ethyl acetate in hexanes); IR (KBr): Vmax 3331,2978,
2951, 1733, 1645, 1619, 1532, 1367, 1319, 1277, 1144,
(KBr): Vmax 3286, 2980, 2936, 1730, 1653, 1619, 1537,
1
1493, 1448, 1349, 1314, 1159, 1131, 1092 cm
;
1H
NMR (500 MHz, CDCl3): d 8.56 (dd, 4J(1H-1H)=
2.5 Hz, 4J(1H-19F)=7.5 Hz, 1H, Ar), 8.12 (ddd, 4J
(1H-1H)=2.5 Hz, 4J (1H-19F)=4.0 Hz, 3J (1H-1H)=
9.0 Hz, 1H, Ar), 7.84 (d, J=7.5 Hz, 2H, Ph), 7.58 (d,
J=7.5 Hz, 1H, Ph), 7.50 (t, J=7.5 Hz, 2H, m-Ar), 7.34
(dd, 3J (1H-1H)=9.0 Hz, 3J (1H-19F)=10.0 Hz, Ar), 7.13
(t, J=5.5 Hz, 1H, (C=O)NH), 5.89 (d, J=8.0 Hz, 1H,
CHNHSO2Ph), 3.97±3.92 (m, 2H, CHH, CHCH2),
t
1
3.60±3.54 (m, 1H, CHH), 1.29 (s, 9H, Bu); 13C NMR
1051, 1025 cm 1; H NMR (500 MHz, CDCl3): d 11.43
(125 MHz, CDCl3): d 168.1, 164.5, 158.1, 156.0, 138.6,
134.2, 134.1, 133.2, 130.9, 129.2, 127.2, 125.6, 118.9,
118.7, 84.2, 55.8, 42.5, 27.6; FAB-HRMS (M+Cs+)
calcd 600.0217, found 600.0195.
(bs, 1H, (C=N)NH (C=O)), 8.76 (bt, J=5.5 Hz, 1H,
CH2NH(C=N)), 8.32 (d, J=2.5 Hz, 1H, Ar), 8.00 (dd,
J=2.5, 9.0 Hz, 1H, Ar), 7.85±7.84 (m, 2H, Ph), 7.58 (t,
J=8.0 Hz, 1H, Ph), 7.49 (t, J=8.0 Hz, 2H, Ph), 7.20 (d,
J=9.0 Hz, 1H, Ar), 6.95 (t, J=8.0 Hz, 1H, NHCO),
5.84 (d, J=7.5 Hz, 1H, HNSO2), 4.28 (t, J=5.5 Hz, 2H,
CH2O), 3.96±3.87 (m, superimposed, 4H, CHCH2,
CH2NH(C=N)), 3.60±3.54 (m, 1H, CHCH2), 1.50 (s,
9H, Bu), 1.48 (s, 9H, Bu), 1.28 (s, 9H, Bu); 13C NMR
(125 MHz, CDCl3): d 168.2, 165.0, 163.3, 156.5, 154.5,
154.1, 150.9, 139.4, 138.8, 133.1, 132.7, 129.2, 127.2,
126.5, 125.0, 114.5, 84.0, 83.4, 68.1, 55.9, 42.4, 39.4,
28.3, 28.0, 27.6; FAB-HRMS (M+Cs+) calcd
883.1949, found 883.1970.
Compound 59. Compound 59 was prepared by the same
procedure as for compound 58. Yield (985 mg, 96%) as
an o€-white solid. Rf=0.24 (silica gel, 50% ethyl acetate
in hexanes); IR (KBr): Vmax 3395, 3297, 3083, 2981,
2937, 1734, 1671, 1620, 1534, 1494, 1460, 1345, 1264,
t
t
t
1156, 1128, 1079, 977, 918, 833, 750, 661, 617, 549,
1
479 cm
;
1H NMR (500 MHz, CDCl3): d 8.46 (dd,
4J(1H-1H)=2.5 Hz, 4J(1H-19F)=7.0 Hz, 1H, Ar), 8.38
(d, J=2.0 Hz, 1H, naphthyl), 8.02 (ddd, 4J(1H-1H)=
2.5 Hz, 4J(1H-19F)=4.0 Hz, 3J(1H-1H)=8.8 Hz, 1H,
Ar), 7.90 (bd, superimposed, J=8.5 Hz, 1H, naphthyl),
7.88 (bd, superimposed, J=8.5 Hz, 1H, naphthyl),
7.83 (bd, J=8.0 Hz, 1H, naphthyl), 7.79 (dd, J=2.0,
8.0 Hz, 1H, naphthyl), 7.62 (ddd, J=1.0, 7.5, 8.5 Hz,
1H, naphthyl), 7.58 (ddd, J=1.0, 7.5, 8.5 Hz, 1H,
naphthyl), 7.27 (br, dd, J=6.0, 8.5 Hz, 1H, CONH),
7.18 (bdd, 3J(1H-1H)=9.0 Hz, 3J(1H-19F)=10.0 Hz, 1H,
Ar), 6.15 (d, J=8.0 Hz, 1H, NHSO2), 4.06 (ddd, J=4.0,
5.5, 8.0 Hz, 1H, CHCH2), 3.93 (ddd, J=4.0, 6.0,
11.0 Hz, 1H, CHCHH), 3.57 (ddd, J=5.5 Hz, 8.5 Hz,
Compound 11. To a solution of 60 (30.0 mg, 0.04 mmol)
in CH2Cl2 (2 mL) tri¯uoroacetic acid (2 mL) was added
dropwise. The reaction mixture was stirred at 25 ꢀC for
2 h. The solvents were removed under reduced pressure
and the residue was puri®ed by RP-HPLC (C-18) to give
11 as yellowish solid (22.0 mg, 92%). tR=12.2 min; IR
(KBr): Vmax 3418, 1679, 1529, 1433, 1354, 1319, 1278,
1198, 1161, 1092, 1046, 932, 837, 802, 756, 688 cm 1; 1H
NMR (500 MHz, methanol-d4): d 8.26 (d, J=2.0 Hz,
1H, Ar), 8.04 (dd, J=2.0, 8.5 Hz, 1H, Ar), 7.82±7.80 (m,
2H, Ph), 7.47±7.36 (m, 4H, Ar), 4.35 (t, J=5.0 Hz, 2H,
OCH2), 4.19 (dd, J=4.0, 14.0 Hz, 1H, CHCH2), 3.75
(dd, J=4.0, 14.0 Hz, 1H, CHCHH), 3.68 (t, J=5.0 Hz,
2H, CH2NH(C=N)), 3.47 (dd, J=11.0, 14.0 Hz, 1H,
t
1H, CHCHH), 1.17 (s, 9H, Bu); 13C NMR (125 MHz,
CDCl3): d 168.3, 164.3, 158.0, 155.8, 135.5, 134.7,
134.1, 134.0, 131.8, 130.5, 129.5, 129.1, 128.6, 127.7,
125.4, 122.0, 118.6, 118.4, 83.9, 55.9, 42.3, 27.4;

1202
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
CHCHH); 13C NMR (125 MHz, methanol-d4): d 167.7,
155.0, 142.2, 140.7, 134.5, 133.6, 130.0, 128.2, 125.9,
115.7, 69.8, 43.3, 41.8, 25.2; FAB-HRMS (M+H+)
calcd 495.1298, found 495.1311.
syringe. After 12 h at room temperature the reaction was
stopped by addition of water (10 mL) and diluted with
ethyl acetate. After phase seperation the aqueous phase
was extracted with ethyl acetate (3Â25 mL). The com-
bined organic extracts were washed succesively with
water (2Â10 mL) and brine (10 mL) and dried over
MgSO4. After ®ltration and evaporation under reduced
pressure the residue was puri®ed by ¯ash column chro-
matography (silica gel, 40% ethyl acetate in hexanes) to
give 62 as a yellowish foam (35 mg, 23%). Rf=0.32
(silica gel, 40% ethyl acetate in hexanes); 1H NMR
Compound 61. To a solution of 58 (100 mg, 0.20 mmol)
in DMF (10 mL) was added 53 (68 mg, 0.22 mmol).
After stirring at room temperature for 4 h, the reaction
mixture was diluted with water (10 mL) and the aqueous
phase extracted with ethyl acetate (3Â10 mL). The
combined organic extracts were washed succesively with
water (2Â10 mL) and brine (20 mL) and dried over
Na2SO4. After ®ltration and evaporation under reduced
pressure the residue was puri®ed by ¯ash column chro-
matography (silica gel, 50% ethyl acetate in hexanes) to
give 61 as a yellowish foam (110 mg, 73%). Rf=0.48
(silica gel, 60% ethyl acetate in hexanes); IR (®lm): Vmax
3318, 2925, 1723, 1623, 1517, 1412, 1324, 1158 cm 1; 1H
(500 MHz, CDCl3):
d 11.45 (bs, 1H, (C=N)NH
(C=O)), 8.65 (bt, superimposed, J=4.5 Hz, 1H,
CH2NH(C=N)), 8.63 (d, superimposed, J=2.0 Hz, 1H,
Ar), 8.22 (d, J=8.5 Hz, 1H, Ar), 8.15 (dd, J=2.0,
8.5 Hz, 2H, Ar), 7.84 (d, J=8.0 Hz, 2H, Ph), 7.56 (t,
J=7.5 Hz, 1H, Ph), 7.48 (bdd, J=7.0, 8.0 Hz, 2H, Ph),
6.98 (t, J=5.5 Hz, 1H, CONH), 5.72 (d, J=7.0 Hz, 1H,
NHSO2), 3.99±3.93 (bm, 1H, CHCH2), 3.88 (ddd,
J=4.5, 5.5, 13.5 Hz, 1H, CHCHH), 3.70±3.58 (m, 3H,
CHCHH, CH2NH(C=N)), 3.26 (t, J=8.0 Hz, 2H,
NMR (500 MHz, CDCl3):
d
11.47 (bs, 1H,
(C=N)NH(C=O)), 8.59 (d, J=2.5 Hz, 1H, Ar), 8.56 (t,
J=10.0 Hz, 1H, NH), 8.43 (t, J=10.0 Hz, 1H, NH),
7.93 (dd, J=2.5 Hz, 10.0 Hz, 1H, Ar), 7.84 (d,
J=9.0 Hz, 2H, Ph), 7.53 (t, J=10.0 Hz, 1H, Ph), 7.46 (t,
J=10.0 Hz, 2H, Ph), 7.21 (d, J=10.0 Hz, 1H, Ar), 6.81
(t, J=10.0 Hz, 1H, CONH), 5.87 (d, J=10.0 Hz, 1H,
NHSO2), 3.98±3.93 (m, 1H, CHCH2), 3.87±3.82 (m, 1H,
CHCHH), 3.72±3.55 (m, 5H, CHCHH, NHCH2,
t
t
SCH2), 1.57 (s, 9H, Bu), 1.50 (s, 9H, Bu), 1.30 (s, 9H,
tBu); 13C NMR (125 MHz, CDCl3): d 168.2, 165.1,
163.4, 156.3, 153.1, 145.3, 141.0, 138.9, 133.1, 132.3,
130.3, 129.2, 127.7, 127.2, 124.7, 84.0, 83.5, 79.6, 55.9,
42.4, 39.2, 30.1, 28.4, 28.0, 27.6; FAB-HRMS
(M+Cs+) calcd 899.1720, found 899.1753.
t
t
CH2NH(C=N)), 1.52 (s, 9H, Bu), 1.47 (s, 9H, Bu),
1.27 (s, 9H, tBu); 13C NMR (125 MHz, CDCl3): d 168.4,
165.7, 163.3, 156.6, 153.2, 146.9, 139.1, 134.7, 133.0,
131.4, 129.1, 127.2, 126.2, 121.0, 114.4, 83.7, 83.5, 79.5,
56.2, 42.4, 42.2, 39.0, 28.3, 28.0, 27.6; FAB-HRMS
(M+Cs+) calcd 882.2109, found 882.2129.
Compound 15. Compound 15 was prepared by the same
procedure as for 11. Yield: (23.0 mg, 92%) as a yellow
solid. tR=16.0 min; H NMR (500 MHz, methanol-d4):
1
d 8.58 (d, J=2.0 Hz, 1H, Ar), 8.06 (dd, J=2.0, 8.5 Hz,
1H, Ar), 7.82 (d, J=7.0 Hz, 2H, Ph), 7.71 (d, J=8.5 Hz,
1H, Ph), 7.48±7.41 (m, 3H, Ar), 4.23 (dd, J=5.0, 9.0 Hz,
1H, CHCH2), 3.80±3.76 (m, 1H, CHCHH), 3.56 (t,
J=6.5 Hz, 2H, CH2NH(C=N)), 3.50±3.43 (m, 1H,
CHCHH), 3.34 (t, J=6.5 Hz, 2H, SCH2); 13C NMR
(125 MHz, methanol-d4): d 167.6, 155.2, 142.1, 140.7,
133.5, 133.3, 132.5, 128.3, 128.0, 126.0, 43.4, 40.7,
32.3; FAB-HRMS(M+H+) calcd 511.1070, found
511.1058.
Compound 14. Compound 14 was prepared by the same
procedure as 11. Yield (33.2 mg, 92%) as a yellow solid.
tR=15.9 min; IR (KBr): Vmax 3364, 3245, 2998, 2584,
1669, 1624, 1555, 1520, 1433, 1313, 1198, 1161, 923, 756,
1
722 cm 1; H NMR (500 MHz, methanol-d4): d 8.61 (d,
J=2.5 Hz, 1H, Ar), 7.92 (dd, J=2.5, 9.0 Hz, 1H, Ar),
7.81 (d, J=7.0 Hz, 2H, Ph), 7.47±7.40 (m, 3H, Ph), 7.11
( d, J=9.0 Hz, 1H, Ar), 4.19 (dd, J=5.0, 9.0 Hz, 1H,
CHCH2), 3.72 (dd, J=5.0, 13.5 Hz, 1H, CHCHH), 3.67
(t, J=6.0 Hz, 2H, NHCH2), 3.52 (t, J=6.0 Hz, 2H,
NHCH2), 3.46 (dd, J=9.0, 13.5 Hz, 1H, CHCHH); 13C
NMR (125 MHz, methanol-d4): d 168.3, 159.0, 148.0,
142.1, 135.8, 133.6, 132.9, 130.0, 127.8, 125.0, 122.3,
114.8 43.2, 42.5, 41.1, 31.1; FAB-HRMS (M+H+)
calcd 494.1458, found 494.1444.
Compound 65. To a solution of 58 (100 mg, 0.20 mmol)
in DMF (10 mL) was added 52 (68 mg, 0.22 mmol).
After 6 h, the reaction mixture was diluted with water
(25 mL) and ethyl acetate. After phase separation the
aqueous phase was extracted with ethyl acetate
(3Â30 mL). The combined organic extracts were washed
succesively with water (2Â20 mL) and brine (20 mL) and
dried over Na2SO4 After ®ltration and evaporation
under reduced pressure the residue was puri®ed by ¯ash
column chromatography (silica gel, 40!50% ethyl
acetate in hexanes) to give 65 as a yellowish foam
(120 mg, 83%). Rf=0.30 (silica gel, 40% ethyl acetate in
Compound 62. To a solution of 54 (1.60 mg, 5.0 mmol)
in THF (50 mL) was added NaH (60% suspension in
mineral oil) (200 mg, 5.00 mmol) at 0 ꢀC. After 15 min
the resulting thiolate solution was ready for use. To a
solution of 58 (100 mg, 0.20 mmol) in DMF (10 mL)
was added the thiolate solution (5.0 mL, 0.5 mmol) by
hexanes); IR (®lm): Vmax 3266, 2977, 1743, 1621, 1520,
1
1451, 1367, 1304, 1158, 1130, 1094, 1014 cm
;
1H

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1203
NMR (500 MHz, CDCl3): d 10.22 (bs, 1H, (C=N)
HN(C=O)), 8.29 (d, J=2.5 Hz, 1H, Ar), 7.92 (dd,
J=2.5, 9.0 Hz, 1H, Ar), 7.85±7.84 (m, 2H, Ph), 7.83±
7.82 (m, 1H), 7.56 (t, J=7.0 Hz, 1H, Ph), 7.48 (t,
J=7.0 Hz, 2H, Ar), 7.08 (d, J=9.0 Hz, 1H, Ar), 6.93 (t,
J=6.0 Hz, 1H, CO(NH)), 5.85 (d, J=8.0 Hz, 1H,
NHSO2), 3.96±3.87 (m, 2H, CHCH2, CHCHH), 3.80±
3.70 (bm, 4H, NH(CH2)2), 3.59±3.54 (m, 1H, CHCHH),
3.23±3.21 (m, 4H, N(CH2)2), 1.48 (s, 18 H, tBu), 1.26 (s,
J=8.5 Hz, 1H, Ar), 7.83 (d, J=8.5 Hz, 1H, naphthyl),
7.79 (dd, J=2.0, 8.5 Hz, 1H, naphthyl), 7.64±7.55
(2Âbr, dd, 2H, naphthyl), 7.07 (d, J=8.5 Hz, 1H, Ar),
6.90 (dd, J=5.5, 5.5 Hz, 1H, CH2NH(C=O)), 5.95 (d,
J=7.5 Hz, 1H, NHSO2), 4.22 (t, J=5.3 Hz, 2H,
OCH2), 4.02 (ddd, J=4.0, 7.5, 8.5 Hz, 1H, CHCH2),
3.93±3.83 (m, superimposed, 3H, CHCHH, CH2NH
(C=N)), 3.55 (ddd, J=5.5, 8.5, 13.5 Hz, 1H, CHCHH),
1.50 (s, 9H, tBu), 1.47 (s, 9H, tBu), 1.17 (s, 9H, tBu); 13
C
t
9H, Bu); 13C NMR (500 MHz, CDCl3): d 168.3, 165.3,
NMR (125 MHz, CDCl3): d 168.3, 164.9, 156.4, 154.0,
152.9, 139.3, 135.6, 134.9, 132.6, 132.0, 129.6, 129.2,
129.1, 128.7, 127.8, 127.7, 127.6, 126.3, 125.0, 122.1,
114.4, 84.0, 83.5, 68.0, 56.1, 42.3, 39.5, 28.3, 28.0,
27.5; FAB-HRMS (M+Cs+) calcd 933.2105, found
933.2116
155.2, 147.5 140.9, 138.8, 133.1, 132.0, 129.2, 127.2,
126.3, 126.0, 119.9, 84.0, 56.0, 50.3, 42.2, 28.1, 27.6;
FAB-HRMS (M+Cs+) calcd 908.2265, found
908.2233.
Compound 16. Compound 16 was prepared by the same
procedure as for 11. Yield: (15.0 mg, 93%) as a yellow-
ish solid. tR=15.3 min; IR (KBr): Vmax 3367, 3239,
Compound 17. Compound 17 was prepared by the same
procedure as for 11. Yield: (32.7 mg, 99%) as an o€-
white to brownish solid. tR=14.5 min. IR (KBr): Vmax
3421, 2999, 2898, 1657, 1635, 1528, 1383, 1351, 1322,
1276, 1198, 1157, 1132, 1080, 1046, 979, 823, 754, 660,
2925, 2857, 1662, 1613, 1523, 1449, 1388, 1320, 1199,
;
1H NMR
1
1173, 1135, 1093, 992, 837, 802, 721 cm
(500 MHz, methanol-d4): d 8.22 (d, J=2.0 Hz, 1H, Ar),
7.95 (dd, J=2.0, 8.5 Hz, 1H, Ar), 7.79 (m, 2H, Ph),
7.47±7.38 (m, 3H, Ph), 7.32 (d, J=8.5 Hz, 1H, Ar), 4.21
(dd, J=5.0, 9.0 Hz, 1H, CHCH2), 3.74 (dd, J=5.0,
10.0 Hz, 1H, CHCHH), 3.67±3.65 (m, 4H, N(CH2)2),
3.49±3.44 (m, 1H, CHCHH), 3.31±3.29 (m, 4H,
N(CH2)2); 13C NMR (125 MHz, methanol-d4): d 172.6,
167.9, 158.4, 148.3, 142.8, 142.2, 129.9, 56.6, 50.9, 46.3,
43.3; FAB-HRMS (M+H+) calcd 520.1614, found
520.1630.
1
550 cm 1; H NMR (500 MHz, methanol-d4): d 8.15 (s,
1H, naphthyl), 7.93 (d, J=2.0 Hz, 1H, Ar), 7.72 (d,
J=8.0 Hz, 1H, naphthyl), 7.69±7.58 (m, 4H, naphthyl,
Ar), 7.42±7.35 (2Âddd, superimposed, 2H, naphthyl),
6.92 (d, J=9.0 Hz, 1H, Ar), 4.21 (dd, J=4.5, 9.8 Hz,
1H, CHCH2), 4.13 (t, J=5.0 Hz, 2H, OCH2), 3.61 (dd,
J=4.5, 13.5 Hz, 1H, CHCHH), 3.57 (t, J=4.5 Hz, 2H,
CH2NH(C=N)), 3.28 (dd, J=9.8 Hz, 13.5 Hz, 1H,
CHCHH); 13C NMR (125 MHz, methanol-d4): d 172.8,
167.1, 159.3, 154.9, 140.1, 139.5, 135.9, 134.1, 133.4,
130.3, 130.2, 129.5, 128.8, 128.7, 128.4, 127.3, 125.7,
123.5, 115.3, 69.7, 56.8, 43.1, 41.8; FAB-HRMS
(M+H+) calcd 545.1455, found 545.1471.
Compound 63. To a solution of 51 (130 mg, 0.43 mmol)
in THF (5.0 mL) was added NaH (60% suspension in
mineral oil) (70 mg, 1.74 mmol) at 0 ꢀC. Stirring was
continued at this temperature for additional 15 min and
the resulting grey suspension was ready for use. To a
solution of 59 (200 mg, 0.39 mmol) in DMF (20 mL) was
added the alkoxide (2.5 mL). After stirring for 1 h at
0 ꢀC the remaining 2.5 mL of the alkoxide was added.
After 3 h at 0 ꢀC the reaction was stopped by addition of
saturated aqueous NH4Cl-solution (10 mL) and diluted
with ethyl acetate. After phase separation the aqueous
phase was extracted with ethyl acetate (3Â25 mL). The
combined organic extracts were washed succesively with
H2O (2Â10 mL) and brine (10 mL) and dried over
MgSO4. After ®ltration and evaporation under reduced
pressure the residue was puri®ed by ¯ash column chro-
matography (silica gel, 50% ethyl acetate in hexanes) to
give 63 as a yellow solid (211 mg, 69%). Rf=0.16 (silica
gel, 50% ethyl acetate in hexanes); IR (KBr): Vmax 3330,
2979, 2934, 1728, 1620, 1570, 1531, 1416, 1329, 1281,
Compound 64. To a solution of 59 (50 mg, 0.10 mmol) in
1-methyl-2-pyrrolidinone (1 mL) was added 53 (58 mg,
0.19 mmol) at room temperature. After 4 h, the reaction
mixture was diluted with water (10 mL) and the aqueous
phase extracted with ethyl acetate (3Â10 mL). The
combined organic extracts were washed succesively with
water (2Â5 mL) and brine (5 mL) and dried over
MgSO4. After ®ltration and evaporation under reduced
pressure the residue was puri®ed by ¯ash column chro-
matography (silica gel, 50% ethyl acetate in hexanes) to
give 64 as a yellow solid (76 mg, 99%). Rf=0.22 (silica
gel, 50% ethyl acetate in hexanes); IR (KBr): Vmax 3300,
3065, 2975, 2931, 1734, 1660, 1620, 1535, 1497, 1347,
1261, 1159, 1129, 1079, 972, 917, 817, 751, 661, 551,
477 cm 1; 1H NMR (500 MHz, CDCl3): d 11.49 (bs, 1H,
(C=N)NH(C=O)), 8.58 (bt, 1H, CH2NH(C=N)), 8.47
(d, J=2.0 Hz, 1H, Ar), 8.41 (dd, J=5.5 Hz, 1H,
NHCH2), 8.37 (d, J=2.0 Hz, 1H, naphthyl), 7.90±7.84
(m, superimposed, 3H, naphthyl), 7.86 (dd, J=2.0,
9.0 Hz, 1H, Ar), 7.79 (dd, J=2.0, 8.5 Hz, 1H, naphthyl),
7.59 (ddd, J=1.5, 7.0, 7.0 Hz, 1H, naphthyl), 7.54 (ddd,
1156, 1079, 1023, 970, 916, 816, 753, 660, 618, 552,
;
1H NMR (500 MHz, CDCl3): d 11.45 (bs,
1
477 cm
1H, (C=N)NH(C=O)), 8.78 (bt, 1H, CH2NH(C=N)),
8.37 (s, 1H, Ar), 8.26 (d, J=2.0 Hz, 1H, naphthyl),
7.92±7.87 (2Âd, J=8.0 Hz, 2H, naphthyl), 7.90 (d,

1204
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
J=1.5, 7.0, 7.0 Hz, 1H, naphthyl), 7.12 (d, J=9.0 Hz,
1H, Ar), 6.69 (t, J=5.5 Hz, 1H, CH2NH(C=O)), 5.89
(d, J=8.0 Hz, 1H, NHSO2), 4.03 (ddd, J=4.0, 8.0,
8.5 Hz, 1H, CHCH2), 3.85 (ddd, J=4.0, 6.0, 14.0 Hz,
1H, CHCHH), 3.70 (bdt, J=5.5, 5.5 Hz, 2H, NHCH2),
3.61±3.50 (m, 3H, CHCHH, CH2NH(C=N)), 1.52 (s,
9H, Bu), 1.47 (s, 9H, Bu), 1.18 (s, 9H, Bu); 13C NMR
(125 MHz, CDCl3): d 168.5, 165.6, 156.6, 153.2, 146.9,
136.0, 134.8, 134.6, 132.0, 131.3, 129.6, 129.2, 128.9,
128.6, 127.8, 127.6, 126.0, 122.2, 120.8, 114.3, 83.8, 83.6,
56.4, 42.4, 42.2, 39.1, 28.3, 28.0, 27.6; FAB-HRMS
(M+Cs+) calcd 932.2265, found 932.2285.
(2Âbddd, superimposed, 2H, naphthyl), 7.13 (bm, 1H,
NH(C=O)), 6.98 (d, J=9.0 Hz, 1H, Ar), 4.01 (ddd,
J=3.5, 8.0, 9.0 Hz, 1H, CHCH2), 3.88 (ddd, J=4.0, 6.0,
13.5 Hz, 1H, CHCHH), 3.73 (bm, 4H, NCH2), 3.55
(ddd, J=5.5, 8.5, 13.5 Hz, 1 H CHCHH), 3.18 (bm, 4H,
NCH2), 1.48 (s, 18 H, tBu), 1.15 (s, 9H, tBu); 13C NMR
(125 MHz, CDCl3): d 168.6, 165.1, 155.2, 147.3, 140.9,
135.6, 134.9, 132.0, 129.6, 129.2, 129.0, 128.7, 127.8,
127.7, 126.2, 126.0, 122.1, 119.8, 119.7, 83.9, 56.1, 50.3,
42.2, 28.2, 28.0, 27.3; FAB-HRMS (M+Cs+) calcd
958.2422, found 958.2458.
t
t
t
Compound 19. Compound 19 was prepared by the same
procedure as for 11. Yield (31.9 mg, 93%) as a yellowish
solid. tR=11.1 min; IR (KBr): Vmax 3401, 3297, 3251,
2996, 2928, 1659, 1613, 1523, 1451, 1385, 1323, 1199,
Compound 18. Compound 18 was prepared by the
same procedure as 11. Yield (81.7 mg, 90%) as an
orange yellow solid. tR=12.4 min; IR (KBr): Vmax
3364, 1676, 1624, 1556, 1520, 1426, 1315, 1241, 1200,
1
1157, 1138, 1078, 992, 808, 753, 720, 660, 549 cm 1; H
1158, 1133, 1076, 1025, 999, 824, 757, 719, 660, 549,
;
1H NMR (500 MHz, methanol-d4): d 8.17
NMR (500 MHz, methanol-d4): d 8.19 (bs, 1H, naph-
thyl), 7.93 (d, J=2.0 Hz, 1H, Ar), 7.76 (bd, J=9.0 Hz,
1H, naphthyl), 7.71±7.61 (m, superimposed, 3H, naph-
thyl, Ar), 7.55 (dd, J=2.0 Hz, 8.8 Hz, 1H, naphthyl),
7.44±7.36 (2Âddd, superimposed, 2H, naphthyl), 6.91
(d, J=8.5 Hz, 1H, Ar), 4.21 (dd, J=4.5, 9.5 Hz, 1H,
CHCH2), 3.61 (dd, J=4.5, 13.5 Hz, 1H, CHCHH), 3.55
(m, 4H, NCH2), 3.29 (dd, J=9.5, 13.5 Hz, 1H,
CHCHH), 3.15±3.09 (m, 4H, NCH2); 13C NMR
(125 MHz, methanol-d4): d 167.6, 158.5, 148.2, 142.1,
139.4, 136.0, 133.5, 133.3, 130.4, 130.3, 129.7, 128.9,
128.5, 127.3, 126.7, 123.6, 121.2, 50.9, 49.6, 48.6,
46.4; FAB-HRMS (M+Cs+) calcd 702.0747, found
702.0784.
1
479 cm
(bs, 1H, naphthyl), 8.14 (d, J=2.0 Hz, 1H, Ar), 7.72
(d, J=8.0 Hz, 1H, naphthyl), 7.68±7.65 (m, 2H, naph-
thyl), 7.57 (bd, superimposed, J=9.5 Hz, 1H, naph-
thyl), 7.55 (dd, superimposed, J=2.0, 9.0 Hz, 1H, Ar),
7.41 (ddd, J=1.5, 7.0, 8.0 Hz, 1H, naphthyl), 7.36
(ddd, J=1.5, 7.0, 8.0 Hz, 1H, naphthyl), 6.74 (d,
J=9.0 Hz, 1H, Ar), 4.25 (dd, J=4.5, 10.0 Hz, 1H,
CHCH2), 3.62 (dd, J=4.5, 14.0 Hz, 1H, CHCHH),
3.52 (t, J=6.0 Hz, 2H, NHCH2), 3.41 (t, J=6.0 Hz,
2H, NHCH2), 3.30 (dd, J=10.0, 14.0 Hz, 1H,
CHCHH); 13C NMR (125 MHz, methanol-d4): d 173.0,
167.8, 147.8, 139.5, 135.9, 135.3, 133.4, 130.3, 130.2,
129.3, 128.7, 128.6, 128.3, 127.2, 123.5, 121.5, 114.5,
57.0, 42.9, 42.5, 41.5; FAB-HRMS (M+Na+) calcd
566.1434, found 566.1453.
Preparation of compounds 20 and 21
Compound 67. To a solution of 57 (0.10 g, 0.20 mmol) in
DMF (8 mL) was added 55 (0.038 g, 0.22 mmol) and
triethylamine (0.06 mL, 0.44 mmol) at room tempera-
ture. After stirring at 25 ꢀC for 16 h, the reaction mix-
ture was diluted with ethyl acetate (10 mL) and water
(10 mL). The layers were seperated and the aqueous
layer was extracted with ethyl acetate (2Â10 mL). The
organic extracts were collected and washed with water
(2Â10 mL) and brine (20 mL) and dried over Na2SO4.
After ®ltration and evaporation under reduced pressure
the residue was puri®ed by ¯ash chromatography (silica,
ethyl acetate) to give 67 as a yellowish solid (110 mg,
92%). Rf=0.43 (silica, ethyl acetate); 1H NMR
(500 MHz, methanol-d4): d 8.57 (d, J=2.0 Hz, 1H, Ar),
7.96 (bm, 1H, Ar), 7.83 (dd, J=2.0, 9.0 Hz, 1H, Ar),
7.80±7.78 (m, 2H, Ar), 7.48±7.39 (m, 4H, Ar), 7.18 (dd,
J=4.0, 6.0 Hz, 2H), 7.06 (d, J=9.0 Hz, 1H, Ar), 4.12
(dd, J=6.0, 8.0 Hz, 1H, CH2CH), 3.89 (t, J=7.0 Hz,
2H, CH2Ar), 3.65 (dd, J=6.0, 14.0 Hz, 1H, CHHCH),
3.46 (dd, J=8.0, 14.0 Hz, 1H, CHHCH), 3.26 (t,
Compound 66. To a solution of 59 (150 mg, 0.29 mmol)
in DMF (5 mL) was added 52 (190 mg, 0.58 mmol) at
room temperature. After 20 h, the reaction mixture was
diluted with water (25 mL) and ethyl acetate. After
phase seperation, the aqueous phase extracted with
ethyl acetate (3Â30 mL). The combined organic extracts
were washed succesively with water (2Â20 mL) and
brine (20 mL) and dried over MgSO4. After ®ltration
and evaporation under reduced pressure the residue was
puri®ed by ¯ash column chromatography (silica gel,
40% ethyl acetate in hexanes) to give 66 as a yellow
solid (240 mg, 99%). Rf=0.33 (silica gel, 50% ethyl
acetate in hexanes); IR (KBr): Vmax 3397, 2979, 2933,
1741, 1610, 1524, 1454, 1367, 1303, 1239, 1157, 1015,
1
975, 834, 752, 661, 615, 552, 477 cm
;
1H NMR
(500 MHz, CDCl3): d 11.49 (bs, 1H, (C=N)NH(C=O),
8.38 (bs, 1H, naphthyl), 8.24 (d, J=2.0 Hz, 1H, Ar),
7.90 (bd, J=7.5 Hz, 1H, naphthyl), 7.87 (bd, J=9.0 Hz,
1H, naphthyl), 7.83 (dd, superimposed, J=2.0, 8.5 Hz,
1H, Ar), 7.82 (d, superimposed, J=8.0 Hz, 1H, naph-
thyl), 7.79 (dd, J=2.0, 8.5 Hz, 1H, naphthyl), 7.64±7.55
t
J=7.0 Hz, 2H, CH2NH), 1.22 (s, 9H, Bu); 13C NMR
(125 MHz, methanol-d4): d 170.4, 168.1, 164.8, 153.6,

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1205
147.9, 142.2, 135.6, 133.6, 132.6, 132.4, 130.1, 128.1,
Preparation of compound 22
127.7, 123.5, 121.8, 114.9, 83.3, 57.3, 43.2, 42.3, 27.9;
Electrospray mass spectrum (M+H+) calcd 609,
observed 609.
Compound 69. To a solution of the ¯uoride 57 (0.10 g,
0.20 mmol) in dry DMF (10 mL) at room temperature a
stream of NH3 (g) was bubbled through for 1 h. After
stirring for 4 h, the reaction mixture was diluted with
ethyl acetate and water. The layers were separated and
the organic layer was washed with water (2Â10 mL),
brine (20 mL) and dried (Na2SO4). The solvent was
removed in vacuo to give 61 as a yellowish oil (0.09 g,
93%). Rf=0.25 (silica, 50% ethyl acetate in hexane); IR
(thin ®lm): Vmax 3359, 1729, 1631, 1516, 1308, 1258,
Compound 20. To a solution of 57 (0.068 g, 0.11 mmol)
in CH2Cl2 (2 mL) was added tri¯uoroacetic acid (2 mL)
at room temperature. After 4 h, the solvent was removed
in vacuo to give an oil which after RP-HPLC (C-18)
gave 20 as a yellow solid (0.056, 97%). 1H NMR
(500 MHz, methanol-d4): d 8.65 (d, J=1.0 Hz, 1H, Ar),
7.93 (dd, J=1.0, 9.0 Hz, 1H, Ar), 7.81 (d, J=8.0 Hz,
2H, Ph), 7.74 (dd, J=3.0, 6.0 Hz, 2H, Ar), 7.58 (dd,
J=3.0, 6.0 Hz, Ar), 7.48 (t, J=7.0 Hz, 1H, Ph), 7.43 (t,
J=8.0 Hz, 2H, Ph), 7.16 (d, J=9.0 Hz, 1H, Ar), 4.20
(dd, J=5.0, 9.0 Hz, 1H, CHCHH), 4.04 (t, J=6.5 Hz,
2H, CH2Ar), 3.74 (dd, J=5.0, 14.0 Hz, 1H, CHCHH),
3.54 (t, J=6.5 Hz, 2H, CH2NH), 3.45 (dd, J=9.0,
14.0 Hz, 1H, CHCHH); 13C NMR (125 MHz, methanol-
d4): d 172.6, 168.1, 152.6, 147.3, 142.0, 135.7, 133.5,
133.1, 132.4, 132.3, 130.0, 127.9, 127.8, 127.4, 122.5,
114.8, 114.6, 56.8, 43.2, 41.2, 27.2; FAB-HRMS
(M+Cs+) calcd 685.0482, found 685.0461.
1
1158, 1093 cm 1; H NMR (500 MHz, methanol-d4): d
8.54 (d, J=2.0 Hz, 1H, Ar), 7.83±7.81 (m, 2H, Ar), 7.74
(dd, J=2.0, 9.0 Hz, 1H, Ar), 7.52±7.43 (m, 3H, Ar), 6.97
(d, J=9.0 Hz, 1H, Ar), 4.12 (dd, J=6.0, 8.0 Hz, 1H,
CH), 3.64 (dd, J=6.0, 13.5 Hz, 1H, CHH), 3.47 (dd,
J=8.0, 13.5 Hz, 1H, CHH), 1.25 (s, 9H, tBu); 13C NMR
(125 MHz, methanol-d4): d 170.5, 168.3, 149.4, 142.2,
134.8, 133.6, 131.7, 130.1, 128.1, 127.1, 122.2, 119.9,
83.4, 57.3, 43.2, 27.9; FAB-HRMS calcd (M+Cs+)
597.0420, found 597.0439.
Compound 70. To a solution of amine 69 (0.23 g,
0.50 mmol) in methanol (15 mL) at room temperature
was added 10% Pd/C (0.10 g) under argon. The ¯ask
was then equipped with a balloon containing H2 (g).
After 8 h, the reaction mixture was ®ltered through a
pad of celite and the solvent removed in vacuo to give
70 as a brownish-reddish oil (0.19 g, 90%). Rf=0.11
(silica, 80% ethyl acetate in hexane); IR (thin ®lm):
Compound 68. To a solution of 57 (0.06 g, 0.13 mmol) in
DMF (10 mL) was added 56 (0.03 g, 0.14 mmol) and
triethylamine (0.04 mL, 0.29 mmol) at room tempera-
ture. After 12 h, the solvent was removed in vacuo to
give 68 as a crude yellow oil (0.09 g, 110%). Rf=0.23
(40% methanol in dichloromethane); 1H NMR
(500 MHz, methanol-d4): d 8.63 (d, J=2.0 Hz, 1H, Ar),
7.93 (dd, J=2.0, 9.0 Hz, 1H, Ar), 7.82 (d, J=6.5 Hz,
2H, Ph), 7.48±7.42 (m, 3H, Ph), 7.09 (d, J=9.0 Hz, 1H,
Ar), 4.08±4.06 (m, 1H, CHCHH), 3.68±3.62 (m, super-
imposed, 5H, NHCH2CH2NH, CHCHH), 3.50±3.44
(m, 1H, CHCHH), 3.30 (t, J=3.5 Hz, 2H, CH2NHAr),
1.24 (s, 9H, tBu); Electrospray mass spectrum calcd
(M+H+) 573, found 573.
Vmax 3360, 2979, 1729, 1625, 1582, 1542, 1508, 1447,
1
1369, 1310, 1248, 1160, 1093, 758, 721, 688, 590 cm
;
1H NMR (500 MHz, methanol-d4): d 7.82±7.80 (m, 2H,
Ar), 7.53±7.49 (m, 1H, Ar), 7.46±7.43 (m, 2H, Ar), 7.11
(d, J=2.0 Hz, 1H, Ar), 7.05 (d, J=2.0 Hz, 1H, Ar), 6.64
(d, J=8.5 Hz, 1H, Ar), 4.08 (dd, J=7.5, 14.5 Hz, 1H,
CH), 3.61 (dd, J=6.0, 13.5 Hz, 1H, CHH), 3.47 (dd,
J=8.0, 13.5 Hz, 1H, CHH), 1.22 (s, 9H, tBu); 13C NMR
(125 MHz, methanol-d4): d 170.8, 170.6, 142.1, 141.2,
134.8, 133.7, 130.1, 128.1, 124.5, 120.6, 116.5, 115.5,
83.3, 57.5, 43.1, 28.0; FAB-HRMS calcd (M+Na+)
435.1702, found 434.1727.
Compound 21. To a solution of 68 (0.09 g, 0.14 mmol) in
CH2Cl2 (5 mL) was added tri¯uoroacetic acid (5 mL) at
room temperature. After 4 h, the solvent was removed in
vacuo to give an oil which after RP-HPLC (C-18) gave
21 as a yellow solid (0.07 g, 83%) tR=14.0 min; 1H
NMR (500 MHz, methanol-d4): d 8.64 (bs, 1H, Ar), 7.94
(d, J=9.0 Hz, 1H, Ar), 7.82 (d, J=7.0 Hz, 2H, Ph), 7.48
(t, J=7.0 Hz, 1H, Ph), 7.43 (t, J=7.0 Hz, 1H, Ph), 7.12
(d, J=9.0 Hz, 1H, Ar), 4.20 (dd, J=5.0, 9.0 Hz, 1H,
CHCH2), 3.73 (dd, J=5.0, 14.0 Hz, 1H, CHCHH),
3.70±3.66 (m, superimposed, 7H, NCH2 CH2N,
CH2N(C=N)), 3.52 (t, J=6.0 Hz, 2H, CH2NHAr), 3.45
(dd, J=9.0, 14.0 Hz, 1H, CHCHH); 13C NMR
(125 MHz, methanol-d4): d 172.7, 168.3, 161.6, 148.0,
142.1, 135.8, 133.5, 132.9, 130.0, 128.0, 127.7, 122.3,
114.9, 56.7, 44.1, 43.2, 42.9, 42.7; FAB-HRMS
(M+H+) calcd 520.1614, found 520.1630.
Compound 71. To a solution of the diamine 70 (0.092 g,
0.20 mmol) in ethanol (20 mL) was added triethylamine
(0.032 mL, 0.22 mmol) and phenyl isothiocyanate
(0.028 mL, 0.22 mmol). After 14 h, the solvent was
removed in vacuo to give a brown residue which was
puri®ed by preparative thin-layer chromatography
(silica, 5% methanol in dichloromethane) to give 71 as a
brown solid (0.082 g, 69%). Rf=0.16 (silica, 5% metha-
nol in dichloromethane); IR (thin ®lm): Vmax 3316, 3061,
2978, 1729, 1624, 1504, 1448, 1368, 1309, 1252, 1159,
1092, 837, 733 cm 1; 1H NMR (500 MHz, CDCl3): d 8.15
(bs, 1H, NH), 7.81 (d, J=7.5 Hz, 2H, Ar), 7.56±7.03

1206
K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
(m, 14 H), 6.78 (bs, 1H, NHC=O), 6.58 (d, J=8.0 Hz,
1H, HNSO2Ph), 4.51 (bs, 1H), 4.05 (bs, 1H), 3.67 (bs,
were harvested, resuspended and washed once in adhe-
sion bu€er (RPMI with 1% BSA, 50 mM HEPES (pH
7.4)) before being allowed to adhere (25,000 cells: 1 h,
37 ꢀC) in the presence of test compounds to the immo-
bilized ligands. Non-adherent cells were removed by
aspiration followed by three rapid washes with adhesion
bu€er and remaining cells were detected using hex-
osaminidase substrate.
t
1
1H), 1.20 (s, 9H, Bu); H NMR (500 MHz, CDCl3): d
180.3, 168.6, 167.4, 147.0, 143.3, 139.6, 137.6, 132.7,
129.0, 128.4, 127.1, 126.6, 125.4, 123.5, 116.1, 83.2, 60.3,
56.5, 42.0, 27.5; FAB-HRMS calcd (M+Cs+) 702.0821,
found 702.0797.
Compound 72. To a solution of the thiourea 71 (0.077 g,
0.14 mmol) in DMF (10 mL) at room temperature was
added triethylamine (0.02 mL, 0.14 mmol) and mercury
(II) chloride (0.04 g, 0.14 mmol). After 4 h, the reaction
mixture was ®ltered through celite and rinsed with ethyl
acetate. The solvent was removed in vacuo to give 72 as
a brown residue (0.05 g, 81%) which was carried onto
the next step. Rf=0.32 (silica, 5% methanol in dichloro-
Chick chorioallantoic membrane (CAM) assay
Animals. Ten day old chick embryos were purchased
from McIntyre Poultry (Lakeside, CA, USA) and incu-
bated at 37 ꢀC with 60% humidity and 51% relative
humidity.
1
methane); H NMR (500 MHz, CDCl3): d 7.98 (m, 2H,
Preparation of ®lter discs. The ®lter discs for placement
on the chick CAM were cut out from Whatman grade 1
®lter paper using a paper punch (6 mm diameter). The
discs were then soaked in 1 mL of a 3.0 mg/mL cortisone
acetate in absolute ethanol solution. The discs were air
dried for 45±60 min in a sterile laminar ¯ow hood.
Ar), 7.53±6.90 (m, 15 H, Ar), 4.16 (m, 1H, CH), 3.83 (bs,
1H, CHH), 3.62 (bs, 1H, CHH), 1.25 (bs, 9H, tBu); FAB-
HRMS calcd (M+Cs+) 668.0944, found 668.0923.
Compound 22. Compound 22 was prepared by the same
procedure as for 10. Yield: (0.04 g, 88%). tR=14.8 min;
1H NMR (500 MHz, methanol-d4): d 7.85±7.82 (m, 3H,
Ar), 7.75 (dd, J=1.5, 8.5 Hz, 1H, Ar), 7.56±7.41 (m, 9H,
Ar), 4.22 (dd, J=5.0, 9.0 Hz, 1H, CH), 3.78 (dd, J=5.0,
13.5 Hz, CHH), 3.48 (dd, J=9.0, 13.5 Hz, CHH); 13C
NMR (150 MHz, methanol-d4): d 171.9, 169.2, 150.7,
141.6, 136.2, 133.1, 131.2, 130.9, 130.6, 129.5, 128.3,
127.5, 124.6, 124.1, 111.9, 111.8, 56.1, 42.8; FAB-HRMS
calcd (M+H+) 480.1342, found 480.1352.
Candling. To locate the optimal position for the place-
ment of the ®lter disc on the CAM and to identify
potential blood vessels for intravenous injections, the
eggs were candled. The broad end of the egg was placed
up to the candlelight to determine the position of the air
sac. To prepare the egg for intravenous injection, the
egg was rotated close to the light to determine the posi-
tion of the prominent blood vessels on the lateral sides
of the egg that were well anchored and close to the sur-
face. The vessel selected for injection was as straight as
possible and from medium to small in size.
Receptor and cell adhesion assays
Human integrins and vitronectin and ®brinogen from
human plasma were prepared as previously detailed;25
aIIbb3 was puri®ed from outdated thrombocytes,26 avb3
and avb5 were from term human placenta.27 The integ-
rins were >95% pure as judged by SDS±PAGE and by
ELISA. Receptor inhibition assays28 and cell attach-
ment25 were performed as previously described.
Dropping the CAM. To separate the CAM from the
shell membrane the broad end of the eggs where the air
sac was located was swabbed with 70% ethanol. A small
hole was drilled through the broad end of the egg.
Another hole, through the shell only, was drilled at the
position where the CAM will separate from the eggshell,
approximately 90ꢀ from the preselected blood vessel.
Curved tip forceps were used to push down on the shell
membrane at the sight of ®lter disc placement. This
pressure allowed the CAM to separate from the shell.
Gentle suction was applied to the hole at the broad end
of the egg which helped create the false air sac directly
over the CAM. To create a window through the eggshell
immediately over the dropped CAM, a 1 cmÂ1 cm box
was drawn directly over the newly created false air sac
and then cut with a model cutting wheel.
Human cell lines and culture and the adhesive char-
acteristics of the cells have been described in full else-
where.25,29 The melanoma cell line M21 attach to
vitronectin predominantly over avb3 with a small con-
tribution from avb5. M21-L lack av expression and were
transfected with full length cDNA of aIIb gene to estab-
lish a line M21-L-IIb30 which adhere to ®brinogen using
expressed cell surface aIIbb3. The carcinoma line UCLA-
P3 expresses avb5 as a vitronectin receptor.25,31
Cell adhesion assays were detailed previously,25,32 limit-
ing integrin usage by selecting the appropriate substrate.
Brie¯y, nontissue culture treated 96-well plates were coated
with puri®ed ®brinogen or vitronectin (5±10 mg/mL). Cells
Cytokine stimulation of angiogenesis. The dried ®lter
discs were coated in 1±2 mL of a 1±2 mg/mL cytokine
solution (bFGF). The cytokine coated ®lter discs were
then placed on their respective CAMS in an area with

K. C. Nicolaou et al./Bioorg. Med. Chem. 6 (1998) 1185±1208
1207
low density of preexisting vessels. The window was
sealed with transparent tape and the embryos were
placed in the incubator.
2. (a) Ojima, I.; Chakravarty, S.; Dong, Q. Bioorg. Med. Chem.
1995, 337. (b) Engleman, V. W.; Kellogg, M. S.; Rogers, T. E.
Ann. Rep. Med. Chem. 1996, 31, 191.
3. (a) Brooks, P. C; Clark, R. A. F.; Cheresh, D. A. Science
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were incubated undisturbed for 18±20 h to facilitate
adherence of ®lter discs to the CAM tissue and to
induce the early stages of angiogenesis. The shell around
the preselected vessel was then removed and a window
was created. A drop of paran oil was placed around
the cut borders of the window to loosen the shell from
the membrane. The shell window was then removed
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volume of the antagonist solution was injected into the
preselected blood vessel. The embryos were then incu-
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Quanti®cation of angiogenesis. In order to quantify
angiogenesis within the chick CAMs, embryos were
removed from the incubator and CAMs harvested.
Using dissecting scissors, the shell immediately sur-
rounding the window was removed to expose the
underlying ®lter disc. Using dissecting forceps and scis-
sors, the portion of CAM tissue containing the cytokine
®lter disc was removed from the rest of the CAM and
placed in a 35Â10 mm sterile petri dish. Each CAM was
individually rinsed with 2 mL of sterile PBS pH 7.40
prior to its analysis. A stereo microscope was used to
count the number of blood vessel branch points directly
beneath the applied ®lter disc. The relative number of
blood vessel branch points is indicative of the number of
new blood vessel sprouts arising from pre-existing blood
vessels. Quanti®cation of the number of branch points
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Acknowledgements
We thank Drs Gary Siuzdak and Dee Huang for their
mass spectroscopy and NMR spectroscopy assistance.
respectively. Financial support by E. Merck (Darm-
stadt), The Skaggs Institute for Chemical Biology, the
National Science Foundation (predoctoral fellowship to
J. I. T.), The Wellcome Trust, London, U.K. (Inter-
national Prize Travelling Research Fellowship to K. C.),
the ALSAM Foundation and the National Institutes of
Health (USA) is gratefully acknowleged. CRGDFV(1)
and CR-bA-DFV(EM-601) were provided by Dr A.
Jonczyk, Department of Chemistry, Merck KGaA.
10. (a) Duggan, M. E.; Fisher, J. E.; Gentile, M. A.; Hartman,
G. D.; Ho€man, W. F.; Hu€, J. R.; Ihle, N. C.; Krause, A. E.;
Leu, T. C.; Nagy, R. M.; Perkins, J. J.; Rodan, G. A.; Rodan,
S. B.; Wesolowski, G.; Whitman, D. B. Abstracts of Papers,
211th ACS National Meeting, New Orleans, LA, March 1996;
MEDI 234. (b) Keenan, R. M.; Miller, W. H.; Kwon, C.; Ali,
F. E.; Callahan, J. F.; Calvo, R. R.; Hwang, S.-M.; Kopple, K.
D.; Peisho€, C. E.; Samanen, J. M.; Wong, A. S.; Yuan, C. K.;
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W.; Graciani, N. R.; Mousa, S. A.; DeGrado, W. F. Bioorg.
Med. Chem. Lett. 1997, 7, 1371. (d) Gadek, T. R.; McDowell,
R. S. Abstracts of Papers, 211th ACS National Meeting, New
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