Synthetic approaches to peptides containing the l-Gln-l-Val-D(S)-Dmt motif

Article abstract of DOI:10.1016/j.bmc.2007.03.005

The pseudoprolines S-Dmo (5,5-dimethyl-4-oxaproline) and R-Dmt (5,5-dimethyl-4-thiaproline) have been used to study the effects of forcing a fully cis conformation in peptides. Synthesis of peptides containing these (which have the same configuration as l-Pro) is straightforward. However, synthesis of peptides containing S-Dmt is difficult, owing to the rapid cyclisation of l-Aaa-S-Dmt amides and esters to form the corresponding diketopiperazines (DKP); thus the intermediacy of l-Aaa-S-Dmt amides and esters must be avoided in the synthetic sequence. Peptides containing the l-Gln-l-Val-D(S)-Dmt motif are particularly difficult, owing to the insolubility of coupling partners containing Gln. Introduction of Gln as N-Boc-pyroglutamate overcame the latter difficulty and the dipeptide active ester BocPygValOC₆F₅ coupled in good yield with S-DmtOH. BocPygVal-S- DmtNH(CH₂)₂C₆H₄NO₂ was converted quantitatively to BocGlnVal-S-DmtNH(CH₂)₂C₆H₄NO₂ with ammonia, demonstrating the utility of this approach. Two peptide derivatives (CbzSerLysLeuGlnVal-S-DmtNH(CH₂)₂C₆H₄NO₂ and CbzSerSerLysLeuGlnVal-S- DmtNH(CH₂)₂C₆H₄NO₂) were assembled, using these new methods of coupling a dipeptide acid active ester with S-DmtOH and introduction of Gln as Pyg, followed by conventional peptide couplings. The presence of the Val caused these peptides to be cleaved very slowly by prostate-specific antigen (PSA) at Leu ↓ Gln, rather than the expected Gln ↓ Val.

Full text of DOI:10.1016/j.bmc.2007.03.005

Bioorganic & Medicinal Chemistry 15 (2007) 3474–3488
Synthetic approaches to peptides containing the
L-Gln-L-Val-D(S)-Dmt motif
Ghadeer A. R. Y. Suaifan,a,ꢀ Tawfiq Arafatb and Michael D. Threadgilla,*
aDepartment of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
bFaculty of Pharmacy & Medicine, University of Petra, Queen Alia Airport Road, 11196 Amman, Jordan
Received 6 November 2006; revised 16 February 2007; accepted 2 March 2007
Available online 6 March 2007
Abstract—The pseudoprolines S-Dmo (5,5-dimethyl-4-oxaproline) and R-Dmt (5,5-dimethyl-4-thiaproline) have been used to study
the effects of forcing a fully cis conformation in peptides. Synthesis of peptides containing these (which have the same configuration
as L-Pro) is straightforward. However, synthesis of peptides containing S-Dmt is difficult, owing to the rapid cyclisation of L-Aaa-S-
Dmt amides and esters to form the corresponding diketopiperazines (DKP); thus the intermediacy of L-Aaa-S-Dmt amides and
esters must be avoided in the synthetic sequence. Peptides containing the L-Gln-L-Val-D(S)-Dmt motif are particularly difficult,
owing to the insolubility of coupling partners containing Gln. Introduction of Gln as N-Boc-pyroglutamate overcame the latter
difficulty and the dipeptide active ester BocPygValOC6F5 coupled in good yield with S-DmtOH. BocPygVal-S-
DmtNH(CH2)2C6H4NO2 was converted quantitatively to BocGlnVal-S-DmtNH(CH2)2C6H4NO2 with ammonia, demonstrating
the utility of this approach. Two peptide derivatives (CbzSerLysLeuGlnVal-S-DmtNH(CH2)2C6H4NO2 and CbzSerSerLysLeuGln-
Val-S- DmtNH(CH2)2C6H4NO2) were assembled, using these new methods of coupling a dipeptide acid active ester with S-DmtOH
and introduction of Gln as Pyg, followed by conventional peptide couplings. The presence of the Val caused these peptides to be
cleaved very slowly by prostate-specific antigen (PSA) at Leu # Gln, rather than the expected Gln # Val.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Sterically demanding pseudoprolines, such as 5,5-
dimethyl-4-oxaproline (Dmo) and 5,5-dimethyl-4-thiapr-
oline (Dmt), have been used to mimic proline in bio-active
peptides to force the conformation of the Aaa-wPro
wholly or largely into the cis tertiary amide conformation.
These peptides have mostly included S-Dmo or R-Dmt,
where the configuration matches that of L-Pro. For
In our previous paper,1 we reported the rapid cyclisation
of L-Val-S-Dmt N-(2-(4-nitrophenyl)ethyl)amide 1 to
give the diketopiperazine 2, expelling the amine 3 with
first-order kinetics in aqueous buffers (t1/2 (pH
8.0) = 15 min, t1/2 (pH 7.0) = 45 min, t1/2 (pH
6.0) = 600 min) (Scheme 1). This dipeptide unit was pro-
posed as a potential molecular clip for use in prodrug
systems. Other dipeptides in this L,S diastereomeric ser-
ies also cyclised rapidly to give diketopiperazines
(DKPs) but the dipeptide derivatives in the L,R series
cyclised much more slowly (for L-Val-R-Dmt N-(2-(4-
nitrophenyl)ethyl)amide: t1/2 (pH 8.0) = 1600 min, t1/2
(pH 7.0) > 2700 min). Thus, in order to build longer
peptides containing this L-Val-S-Dmt feature, the syn-
thetic sequence must not involve generation of L-Val-
S-Dmt amides or esters as intermediates.
O
S
N
NO2
NO2
NH2
O
N
H
1
i
S
O
N
H
+
N
H
2
O H2N
Keywords: Dimethylthiaproline; Pseudoproline; Prostate-specific
antigen; Peptide synthesis; Pyroglutamate.
3
*
Corresponding author. Tel.: +44 1 225 386 840; fax: +44 1 225 386
114; e-mail: m.d.threadgill@bath.ac.uk
Scheme 1. Rapid spontaneous cyclisation of the L-Val-S-Dmt amide 1
to form the DKP 2, expelling the primary amine 3. Reagent and
conditions: (i) aq buffer pH 6.0, pH 7.0 or pH 8.0.
ꢀ
Present address: Faculty of Pharmacy, University of Jordan,
Amman, Jordan.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.005

G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
3475
example, R-Dmt has been incorporated into peptides
mimicking amyloid protein,2 into a neuroprotective tri-
peptide,3 into an oxytocin analogue,4 into a d-conotoxin
analogue,5 into analogues of morphiceptin and endo-
morphin-26 and into an analogue of aspergillamide.7
Interestingly, although the L-Aaa-R-Dmt unit exists in
virtually exclusively the cis-amide conformation,1,4–6,8,9
up to 10% of the trans-amide rotamer is present in the
diastereomeric L-Aaa-S-Dmt series.1 S-Dmo has been
used frequently in peptide synthesis as a masked form
of serine to avoid problems of insolubility and aggrega-
tion during assembly of medium-to-large peptides10–12
and has itself been used to control conformation in pep-
tides.8,13 Owing to the relatively slow cyclisation of L-
amino-acyl-R-Dmt derivatives, it has been possible to
use R-Dmt in conventional peptide synthesis in solution
and on the solid phase.2–6 Indeed, some L-amino-acyl-R-
Dmt amides have been sufficiently stable to allow (pre-
sumably rapid) biochemical and biological evaluation.3,6
Notably, there have been no previous reports of assem-
bly of peptides containing S-Dmt, presumably since
conventional C ! N stepwise synthesis is precluded by
the rapid formation of DKPs from L-amino-acyl-S-
Dmt amides.1
is caused by the tertiary nature of the amide and by
the presence of the buttressing geminal dimethyl unit.
Introduction of the amino-acid as D-Cys and ring-clo-
sure to the thiazolidine late in the synthetic sequence
may obviate this problem. Second, no DKP formation
occurs from L-Val-S-Dmt dipeptides under acidic condi-
tions, as the primary amine is protonated and, hence,
not nucleophilic. Alternatively, it might be expected that
DKP formation could be diminished by diminishing the
electrophilicity of the carbonyl electrophile. This could
potentially be achieved by attempting the couplings of
the required amino-acids with L-Val-S-DmtOH, which,
under the basic conditions of the couplings, should form
a C-terminal carboxylate salt with much reduced elec-
trophilicity. Third, appropriately protected dipeptides
could be coupled with S-DmtOH, although this ap-
proach may lead to problems of loss of stereochemical
integrity in the central amino-acid of the tripeptides
formed.
2.1. Cyclocondensation of pre-formed Aaa-D-CysNHR
peptides
Dmo residues have been used as temporary protection
for Ser and Thr in peptide synthesis and can be con-
structed by treatment of the appropriate Aaa-Ser or
Aaa-Thr carboxylic acid or ester with 2,2-dimethoxy-
propane under acidic conditions.18 Similarly, Aaa-Cys
carboxylic acid and esters can be converted into
Aaa-Dmt.13,19 However, there are no reports of cyclo-
condensation of in-chain Cys residues of the type
-Aaa-Cys-Aaa- to give the corresponding -Aaa-Dmt-
Aaa- peptides. A short study was therefore undertaken
to investigate the feasibility of this process. The first tar-
get as a model substrate for reaction with 2,2-dimeth-
oxypropane was N-benzoyl-L-Cys N-butylamide; this
substrate would test both the feasibility and the regio-
chemistry of the cyclisation (thiazolidine or tetrahydro-
thiazine). However, as shown in Scheme 2, treatment of
L-cystine dimethyl ester 4 with butylamine, followed by
benzoylation, gave the sulfides 5 and 6 in low yields as
In this paper, we report our development of an effective
synthetic route to peptides containing L-Aaa-S-Dmt
units, overcoming issues of poor reactivity and poor
solubility. The sequences Cbz-L-Ser-L-Lys-L-Leu-L-Gln-
L-Val-S-DmtNH(CH2)2C6H4NO2 47 and Cbz-L-Ser-L-
Ser-L-Lys-L-Leu-L-Gln-L-Val-S-DmtNH(CH2)2 C6H4NO2
51 (Scheme 5) were selected as targets for synthesis, as
these were required as part of a study on development
of prodrugs from which active drugs are released by the
peptidase activity of prostate-specific antigen (PSA).
The sequences HisSerSerLysLeuGln # Aaa and SerLy-
sLeuGln # Aaa are cleaved by PSA at the points shown
# and are not substrates for cleavage by other pepti-
dases.14 Peptidases present special problems as activating
enzymes, in terms of prodrug design in general and linker
design in particular. Exopeptidases, particularly carboxy-
peptidases, can release amine-containing drugs directly
from amides at the C-terminal of short peptides.15 How-
ever, some activating enzymes, including PSA, are
endopeptidases, cleaving only amide bonds between
amino-acids. In several cases, this leads to release of a
drug molecule still carrying one or more amino-acyl
unit; this may or may not be deleterious to the desired
pharmacological activity.14,16,17 Our design of a molecu-
lar clip1 to overcome this problem involves placing a
L-Val-S-Dmt unit to the C-terminal side of the point at
which the endopeptidase cuts the peptide; when this is
released by the peptide cleavage, it rapidly forms a DKP
and expels the active drug.
1
the sole identifiable products. The H NMR spectra of
these two compounds were very similar; they were
distinguished principally through the observation of
20
D
an optical rotation ½aꢀ þ 41ꢁ for 5, whereas the more-
polar 6 was optically inactive, as is consistent with its
meso relative stereochemistry. Carrying out the two
reactions in the opposite order, that is, treatment of
N,N0-dibenzoyl-L,L-cystine dimethyl ester 720 with
butylamine at elevated temperature, gave only the opti-
cally active sulfide 5 in poor yield. The mechanism of the
loss of one sulfur atom in these processes is unclear but
the formation of the meso 6 suggests that a planar dehy-
droalanine-like intermediate may be involved. Interest-
ingly, 7 did react conventionally with the alternative
nitrogen nucleophile hydrazine to give the expected
N,N-dibenzoyl-L,L-cystine dihydrazide 8 in good yield.
2. Synthesis of peptides
Three alternatives were explored to avoid the synthetic
pitfall of the rapid cyclisation of L-Val-S-Dmt amides
and esters. First, the rapid formation of the DKP is pro-
moted by the very high proportion of L-Val-S-Dmt
dipeptides in the cis amide conformation; in turn, this
Replacing the N-butylamide in the target with a naphth-
2-ylamide, L,L-cystine N,N-bis(naphth-2-yl)amide 9 was
acetylated to give the bis-acetamide 10 (Scheme 3).
Reductive cleavage of the disulfide with propane-1,3-
dithiol under basic conditions afforded a good yield of

3476
G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
N-terminal amine should be protonated and not react as
-
+
NH3
Cl
MeO2C
NHCOPh
MeO2C
a nucleophile to trigger formation of DKPs. Under basic
conditions, the C-terminal carboxylate would be ex-
pected to be ionised and not be electrophilic. Under
intermediate conditions, an unreactive zwitterion should
be present, rather than a free amine and a carboxylic
acid. To test this concept, an attempt was made to cou-
ple Boc-L-Gln to L-Val-S-Dmt (Scheme 4); this provides
the most severe test of the sequence, as L-Val-S-Dmt N-
(2-(4-nitrophenyl)ethyl)amide cyclises to form the DKP
17 and 2-(4-nitrophenyl)ethylamine with t1/2 = 15 min at
pH 8.0.1 Deprotection of Boc-L-Val-S-DmtOH 151 with
trifluoroacetic acid, followed by reaction of the interme-
diate 16 with Boc-L-GlnOSu, however, gave only the
DKP 2. Thus couplings involving Aaa-Dmt dipeptides
are unlikely to be successful in the L,S relative
configuration.
S
S
S
S
-
+
H3N
Cl
PhCOHN
CO2Me
CO2Me
4
7
i,ii
i
iii
CONHBu
CONHBu
NHCOPh
NHCOPh
S
S
+
PhCOHN
CONHBu
PhCOHN
CONHBu
6
5
H2NHNOC
NHCOPh
2.3. Assembly of target SKLQ-S-Dmt and SSKLQ-S-
Dmt amides through coupling of dipeptides to S-DmtOH
S
S
In the light of the failure to convert Cys to Dmt in a pre-
assembled model peptide and of the formation of DKPs
upon attempted coupling to Aaa-DmtOH dipeptides, it
PhCOHN
CONHNH2
8
Scheme 2. Model experiments to investigate the possibility of forming
the tetrahydrothiazole ring late in the synthetic sequence. Part A.
Reagents: (i) BuNH2; (ii) PhCOCl, NaOAc, H2O, Et2O; (iii)
N2H4ÆH2O, MeOH.
S
N
S
N
CO2H
CO2H
NH3
i,ii
O
O
NHBoc
F3CCO2
N-acetylcysteine N-(naphth-2-yl)amide 11. However,
treatment with 2,2-dimethoxypropane did not lead to
the desired thiazolidine 12; the isolated monothioacetal
13 and the enol thioether 14 arose from reaction at the
sulfur nucleophile only. This result suggested that for-
mation of the 2,2-thiazolidine unit late in the synthetic
sequence was not feasible.
15
16
S
N
S
H
N
O
CO2H
BocGlnVal
O
N
H
Me
Me
2.2. Coupling of Aaa-D-DmtOH
2
17
The second approach towards synthesis of the Dmt pep-
tides was to keep the dipeptide Aaa-Dmt with a carbox-
ylic acid at the C-terminal. Under acidic conditions, the
Scheme 4. Formation of diketopiperazine 2 during attempted peptide
coupling of Val-S-DmtOH 16. Reagents: (i) CF3CO2H; (ii) BocGlnOSu,
Et3N, THF, DMF.
2
S
HS
H
N
H
N
RHN
AcHN
ii
O
O
9: R = H
11
i
10: R = Ac
iii
iii X
MeO
S
S
+
S
H
N
H
N
H
N
N
Ac
AcHN
AcHN
O
O
O
13
14
12
Scheme 3. Model experiments to investigate the possibility of forming the tetrahydrothiazole ring late in the synthetic sequence. Part B. Reagents:
(i) Ac2O, Et3N; (ii) CH2(CH2SH)2, Pri2NEt, THF; (iii) Me2C(OMe)2, TsOH, CH2Cl2.

G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
3477
was necessary to devise a synthetic sequence which
avoided Aaa-Dmt dipeptides with free amines at the
N-terminus. The strategy of attaching dipeptides to
Dmt was developed, as shown in Scheme 5, despite the
usual propensity for loss of stereochemical integrity in
fragment-wise assembly of peptides. As noted previ-
ously,1 attempted couplings to Dmt amides lead to
ring-opening of the thiazolidine, so the C-terminal N-
(2-(4-nitrophenyl)ethyl)amide was introduced later in
the sequence and couplings of dipeptides to S-DmtOH
21 were studied. L-ValOMe 18 was coupled with Boc-
L-Gln by the DCC method to give Boc-L-Gln-L-ValO-
Me 19. Base-hydrolysis of the methyl ester gave the acid
20, which failed to couple with 21 using a variety of
coupling systems, including the acyl fluoride method
which had been successful1 in coupling single amino-
acids to Dmt. To check that acyl fluorides derived from
dipeptides were, indeed, stable and capable of coupling
effectively with amines, L-ValOMe 18 was coupled with
Boc-L-PheOH by the DCC method to give Boc-L-Phe-L-
ValOMe 23. Hydrolysis of the ester and formation of
the acyl fluoride 25 from the dipeptide acid 24, using
cyanuric fluoride, were uneventful. This dipeptide acyl
fluoride reacted efficiently with 2-(4-nitrophenyl)ethyl-
amine 3 to give the dipeptide amide 26, without any
evidence of loss of stereochemical integrity. Thus cou-
plings with dipeptide acyl fluorides are feasible21 but
either the side-chain primary amide of the Gln or the
S
N
O
iii
i
ii
H
CO2H
ValOMe.HCl
BocGlnValOMe
BocHN
X
N
H
CO2H
BocGlnVal
18
iv
19
20
22
CONH2
vii
O
vi
ix
BocPheValOR
BocHN
Ph
F
BocPheValHN
N
H
O
23: R = Me
24: R = H
25
26
NO2
v
CONH2
S
x
O
CO2R
O
N
CO2Bn
BocHN
CO2Bn
xiv
N
H
N
Cl
CO2H
H2
Boc
viii
27: R = H
29
30
21
xi
28: R = Bn
H
N
xii
S
N
CO2R
O
CO2H
O
N
Boc
N
CO2H
H
N
O
Boc
O
N
O
35a: S,S,S
35b: S,R,S
31
32: R = Bn
33: R = H
x
Boc
O
xiii
34: R = C6F5
S
S
N
xiii
H
xv
N
N
H
CO2C6F5
H
N
O
O
N
O
N
N
Boc
O
O
Boc
NO2
O
O
37
36
x
S
N
38: R = BocGlnVal
xvii
xviii
xvii
xix
H
N
39: R = GlnVal F3CCO2H salt
40: R = BocLeuGlnVal
R
41: R = LeuGlnVal F3CCO2H salt
44: R = FmocLys(Boc)LeuGlnVal
45: R = Lys(Boc)LeuGlnVal
O
NO2
xx
46: R = CbzSer(But)Lys(Boc)LeuGlnVal
xxi
xvii
xxii
47: R = CbzSerLysLeuGlnVal F3CCO2H salt
48: R = FmocSer(But)Lys(Boc)LeuGlnVal
49: R = Ser(But)Lys(Boc)LeuGlnVal
xx
xxi
xvii
50: R = CbzSer(But)Ser(But)Lys(Boc)LeuGlnVal
51: R = CbzSerSerLysLeuGlnVal F3CCO2H salt
42: FmocLys(Boc)OH
43: FmocLys(Boc)OC6F5
xvi
Scheme 5. Synthesis of target peptide derivatives containing S-Dmt-NH(CH2)2C6N4NO2. Reagents: (i) BocGlnOH, DCC, HOBt, CH2Cl2; (ii)
NaOH, MeOH; (iii) various coupling conditions (see text); (iv) BocPheOH, DCC, HOBt, CH2Cl2; (v) NaOH, aq MeOH; (vi) cyanuric fluoride,
pyridine; (vii) O2NC6H4(CH2)2NH2ÆHCl, Et3N, DMAP, CH2Cl2; (viii) BnCl, Et3N, THF; (ix) Boc2O, Et3N, DMAP, CH2Cl2; (x) NH3, aq THF; (xi)
H2, Pd/C, MeOH; (xii) DCC, HOBt, CH2Cl2, then ValOBn, Pri2NEt; (xiii) C6F5O2CCF3, pyridine, DMF; (xiv) 21, Pri2NEt, DMF; (xv)
O2NC6H4(CH2)2NH2ÆHCl, Et3N, CH2Cl2; (xvi) C6F5OH, DCC, EtOAc; (xvii) F3CCO2H, CH2Cl2; (xviii) BocLeuOSu, Et3N, DMAP, THF, DMF;
(xix) 43, Et3N, DMAP, THF, DMF; (xx) Et2NH, CH2Cl2; (xxi) CbzSer(But)OSu, Et3N, DMAP, THF, DMF; (xxii) FmocSer(But)OSu, Et3N,
DMAP, THF, DMF.

3478
G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
steric crowding of the secondary amine in Dmt pre-
vented the desired coupling.
Boc-L-LeuOSu (giving 40), deprotection (giving 41) and
coupling with Fmoc-L-Lys(Boc)OPFP 43 (prepared
from Fmoc-L-Lys(Boc)OH 42) to afford the Fmoc-L-
Lys-L-Leu-L-Gln-L-Val-S-Dmt amide 44. The change
from Boc to Fmoc in the main-chain protection strategy
was necessitated by the need for simultaneous acid-
catalysed cleavage of all the side-chain protecting
groups in the target peptides. Cbz is unavailable for pro-
tection, as hydrogenolysis would also reduce the nitro
group and hydrogen bromide is known to be deleterious
to the thiazolidine ring. The Fmoc was removed very
rapidly and efficiently with diethylamine, rather than
the more usual piperidine; this approach using a very
volatile secondary amine greatly facilitated the isolation
of the N-unprotected pentapeptide amide 45.
We have reported1 that S-DmtOH also couples with
Boc-L-Leu pentafluorophenyl (PFP) ester. The coupling
of a suitable dipeptide PFP ester was therefore exam-
ined. Since incorporation of the required Gln had low-
ered yields and impeded coupling reactions owing to
poor solubility of the primary amide in organic solvents,
we sought to introduce the carbon framework of the Gln
in a more lipophilic form. N-Boc pyroglutamates (Boc-
Pygs) are known22,23 to react with carbon nucleophiles
(enolates, Grignard reagents) at the lactam carbonyl;
this system also reacts with primary and heterocyclic
amines to give Nd-substituted Gln derivatives.24 We
rationalised that Boc-Pyg could also act as a lipophilic
synthon for Gln, forming Boc-Gln on treatment with
ammonia. As a model sequence, L-PygOH 27 was con-
verted to its benzyl ester 28 by alkylation of the carbox-
ylate anion with benzyl chloride. Boc was then
introduced to the lactam nitrogen, giving the Boc-L-
Pyg benzyl ester 31. Ring opening of this lactam with
aqueous ammonia in THF gave Boc-L-Gln benzyl ester
30 in very high yield, indicating that this strategy may be
applied to introduction of Gln at the N-terminal of large
and more complex peptides as the more soluble Boc-
Pyg, followed by treatment with ammonia.
Now, simple coupling of 45 with Cbz-L-Ser(But)OSu and
removal of the side-chain protecting groups with trifluo-
roacetic acid gave an excellent yield of the hexapeptide target
CbzSerLysLeuGlnVal-S-DmtNH(CH2)2C6H4NO2 47.
This carries the SerLysLeuGln# minimal sequence
claimed14 to be required for recognition and cleavage by
PSA, with suitable chromophoric markers for the fates
of the N-terminal (Cbz) and C-terminal (nitrophenyl)
products of the enzymic cleavage studies. The corre-
sponding heptapeptide target 51, carrying two serines,
was accessed by coupling of 45 with FmocSerOSu, re-
moval of Fmoc with diethylamine (giving 49), coupling
with CbzSerOSu and exposure of the side-chain func-
tional groups with trifluoroacetic acid.
Taking this tactic forward, hydrogenolysis of the benzyl
ester of 29 gave the carboxylic acid 31, which was cou-
pled with L-Val-OBn via the hydroxybenzotriazole ester,
affording Boc-L-Pyg-L-ValOBn 32. Removal of the ben-
zyl ester protection (giving Boc-L-Pyg-L-ValOH 33) was
followed by activation of the carboxylic acid as the PFP
ester 34, using pentafluorophenyl trifluoroacetate25
which avoided the isolation difficulties sometimes associ-
ated with the formation of PFP esters using DCC. This
active ester acylated the hindered secondary amine of S-
DmtOH 21, giving two isomeric products in 52% total
isolated yield, showing very good efficiency for this type
of coupling.1 These two isomers were shown by 1H
NMR to be the diastereoisomers 35a and 35b (diastereo-
meric ratio 4.2:1); the major isomer was ascribed to be
the LLS isomer 35a with the minor LDS isomer 35b aris-
ing from partial racemisation of the Val a-chiral centre
in the active ester 34 under the basic reaction conditions,
a known problem in assembly of peptides from pre-
formed segments. Conversion of the BocPygValDmt
35a to the PFP active ester 36 and reaction with 3 intro-
duced the model for the amine-containing drug in 37,
without any evidence of racemisation as is usual for such
couplings with Dmt active esters.1 At this point in the
synthetic sequence, the Pyg ring was opened quantita-
tively with ammonia to give the corresponding BocGln
peptide 38. Thus Pyg is shown to be a very effective syn-
thon for Gln, providing suitable solubility in organic
solvents to allow the more challenging couplings to take
place. In the present sequence, the lipophilicity of the
ValDmtNH(CH2)2C6H4NO2 unit provided sufficient
solubility to facilitate the remaining couplings. The fol-
lowing steps used conventional solution-phase stepwise
N-deprotection and active-ester coupling cycles through
acid-catalysed removal of Boc (giving 39), coupling with
2.4. Synthesis of control peptide Cbz-SKLQV-OMe
The peptide amides 47 and 51 were designed as sub-
strates for cleavage by PSA. However, these peptide
constructs are modified in two ways from the construct
SerLysLeuGlnLeu-Drug reported by Denmeade to be
cleaved by this enzyme.14 First and more importantly,
the highly bulky Dmt unit is present at the C-terminus
of the sequence recognised by PSA; second, Leu has
been replaced by the (apparently) very similar Val (lipo-
philic, aliphatic) to the C-terminal side of the expected
cleavage-point in 47 and 51. It was therefore necessary
to prepare and evaluate a control peptide ester 58 in
which the Dmt is absent but the Val is present to eluci-
date which feature may be responsible for any modifica-
tion of the sequence-selectivity of the enzymic cleavage.
This control peptide CbzSerLysLeuGlnValOMe 58 was
assembled largely by conventional stepwise C ! N solu-
tion-phase synthesis, as outlined in Scheme 6. In this se-
quence, it was unnecessary to employ the tactic of
incorporating Gln as Pyg, as there were no difficult
couplings. As before, this target peptide carried Cbz
at the N-terminal.
3. Cleavage with PSA
Samples of the peptides 47, 51 and 58 were incubated
with PSA in aqueous buffer at pH 7.4 at 37 ꢁC and the
compositions of the reaction mixtures were monitored
by HPLC at various time-points for up to 7 d for con-
sumption of the starting peptide constructs. The rates

G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
3479
19: BocGlnValOMe
i
ii
i
iii
iv
47 Cbz-Ser-Lys-Leu-Gln-Val-Dmt-NH(CH2)2C6H4NO2
51 Cbz-Ser-Ser-Lys-Leu-Gln-Val-Dmt-NH(CH2)2C6H4NO2
52: GlnValOMe F3CCO2H salt
53: BocLeuGlnValOMe
54: LeuGlnValOMe F3CCO2H salt
55: FmocLys(Boc)LeuGlnValOMe
56: Lys(Boc)LeuGlnValOMe
i
i
Gln-Val-Dmt-NH(CH2)2C6H4NO2 Val-Dmt-NH(CH2)2C6H4NO2
57: R = CbzSer(But)Lys(Boc)LeuGlnValOMe
58: R = CbzSerLysLeuGlnValOMe F3CCO2H salt
v
i
39
1
S
O
N
Scheme 6. Synthesis of control peptide 58. Reagents: (i) F3CCO2H,
CH2Cl2; (ii) BocLeuOSu, Et3N, DMAP, THF, DMF; (iii) 43, Et3N,
DMAP, THF, DMF; (iv) Et2NH, CH2Cl2; (v) CbzSer(tBu)OSu, Et3N,
DMAP, THF, DMF.
H
2
N
H
O
+
NO2
3
H2N
of cleavage by different batches of the PSA enzyme var-
ied markedly; the results of typical incubation experi-
ments are shown in Figure 1. It is immediately evident
that the peptides 47, 51 and 58 (containing -SerLy-
sLeuGlnVal-) were cleaved very slowly, with half-lives
of several days; this contrasts with the rapid cleavage
of peptides containing -SerLysLeuGlnLeu- under simi-
lar conditions reported by Denmeade et al.14 However,
even though peptides 47, 51 and 58 were poor sub-
strates, was the cleavage point Gln # Val, as predicted
by the results of Denmeade showing cleavage at
Gln # Leu?14 If 47 and 51 were cleaved at Gln # Val,
then L-Val-S-Dmt N-(2-(4-nitrophenyl)ethyl)amide 1
would be released; this dipeptide amide should cyclise
very rapidly (<1 h at pH 7.4) to give the DKP 2 and
2-(4-nitrophenyl)ethylamine 3, as shown in Scheme 7.
Similarly, cleavage of 58 at Gln # Val should release L-
valine methyl ester 18. However, HPLC analysis of the
reaction mixtures from 47 and 51 showed that 2 and 3
were absent but that the sole product containing the
nitrophenyl chromophore had retention time equal to
that of GlnValDmt N-(2-(4-nitrophenyl)ethyl)amide
39. Similarly, GlnValOMe 52 was identified as the prod-
uct of cleavage of 58 by PSA. These data indicate that
not only does replacement of Leu by Val make the pep-
tides poor substrates for PSA-mediated cleavage but
also that the cleavage point is changed from SerLy-
sLeuGln # Leu to SerLysLeu # GlnVal. The cleavage
of the control peptide confirms that the effects of slow-
ing and change in site of the cleavage are not due to
the presence of Dmt.
58 Cbz-Ser-Ser-Lys-Leu-Gln-Val-OMe
i
i
52 Gln-Val-OMe
Val-OMe 18
Scheme 7. Predicted outcomes and actual outcomes of cleavage of
peptide constructs 47, 51 and 58 with PSA. The red arrows indicate the
predicted cleavage points whereas the blue arrows show the actual
cleavage points. Reagent and condition: (i) PSA, aq buffer pH 7.4.
4. Conclusions
In this paper, we have reported the development of an
efficient synthetic route to peptide derivatives containing
L-Val-S-Dmt. Although this dipeptide is of considerable
potential for use as a molecular clip in the design of
endopeptidase-activated prodrugs, its synthesis and
incorporation into longer peptides presents some chal-
lenges owing to the very rapid cyclisation of L-Val-S-
Dmt amides and esters to the corresponding DKP 2.1
First, it proved impossible to carry out a cyclocondensa-
tion with 2,2-dimethoxypropane on a model peptide
containing Cys with the aim of generating the target
peptide containing D-Cys, then converting the latter into
S-Dmt at the end of the sequence. The isolated products
arose from reaction at sulfur only. An interesting desul-
furisation was noted during the preparation of a model
Cys-containing peptide. Second, it was observed that the
drive to cyclisation of L-Val-S-Dmt is so great that even
the unmasked dipeptide 16 (which should not exist in
the cyclisable H2N–CO2H prototropic form) cyclised
to the corresponding DKP 2 before coupling with an
amino-acid active ester could be achieved.
100
80
60
40
20
0
Assembly of the required peptides containing L-Val-
S-Dmt was achieved by coupling of dipeptide acid
pentafluorophenyl esters to S-DmtOH 21. Yields of this
critical coupling were satisfactory but were achieved at
the expense of some loss of stereochemical integrity at
the Val of the Pyg-Val acylating unit. The diastereoiso-
mers were readily separable. Dipeptide acid fluorides
were shown for the first time to be stable and useful in
simple peptide couplings but failed with DmtOH.
0
20
40
60
80 100 120 140 160 180
Time (h)
51
47
58
Figure 1. Time courses of degradation of peptide derivatives
CbzSKLQVDmtNPEA 47, CbzSSKLQVDmtNPEA 51 and
CbzSKLQVOMe 58 by PSA. Data are taken from typical experimen-
tal runs.
Gln could not be employed directly in this sequence
owing to the poor solubility of intermediates containing
this residue in solvents compatible with the formation

3480
G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
of the dipeptide acid pentafluorophenyl ester and with
the coupling reaction. N-Boc-pyroglutamic acid (Boc-
Pyg) was used as a masked form of Gln in the difficult
coupling steps where solubility and ease of chromato-
graphic isolation were essential. Simple treatment of
BocPyg peptides with ammonia gave the corresponding
BocGln peptides in quantitative yields, pointing to
the more general utility of this tactic in incorporating
the poorly soluble and potentially aggregating Gln
residue in ‘difficult’ peptides. A hexapeptide amide
CbzSerLysLeuGlnVal-S-Dmt-NH(CH2)2C6H4NO2 47
and a heptapeptide amide CbzSerSerLysLeuGlnVal-S-
Dmt-NH(CH2)2C6H4NO2 51 were successfully assem-
bled using these methods, together with an appropriate
protecting-groups strategy; thus the L-Val-S-Dmt unit
can be efficiently incorporated into peptides by our new
approach.
H), 3.20 (2H, dd, J = 13.3, 5.1 Hz, 2· b-H), 4.73 (2H,
ddd, J = 9.8, 8.2, 5.1 Hz, 2· a-H), 7.44 (2H, t,
J = 7.4 Hz, 2· Ph 4-H), 7.52 (4H, t, J = 7.4 Hz, 2· Ph
3,5-H2), 7.85 (4H, d, J = 7.4 Hz, 2· 2,6-H2), 8.62 (2H,
d, J = 8.2 Hz, 2· PhCONH), 9.37 (2H, br, 2· NHNH2);
MS m/z 477.1370 (M+H) (C20H25N6O2S2 requires
477.1379).
5.3. R,R-Bis(2-benzoylamino-3-butylamino-3-oxopro-
pyl)sulfide (5) and R,S-bis(2-benzoylamino-3-butylamino-
3-oxopropyl)sulfide (6)
Cystine dimethyl ester dihydrochloride
4
(5.0 g,
15 mmol) was dissolved in BuNH2 (100 mL) at
ꢂ78 ꢁC. The solution was heated under reflux for 5 h.
The evaporation residue, in MeOH, was acidified
(HCl). Evaporation afforded crude cystine N,N0-dibutyl
amide (14.8 g) as a yellow viscous oil: MS m/z 351.1874
(M+H) (C14H31N4O2S2 requires 351.1888). This mate-
rial was stirred with NaOAc (10 g) in H2O (100 mL)
and Et2O (100 mL). Benzoyl chloride (9.1 g, 65 mmol)
was added at 0 ꢁC and the mixture was stirred vigor-
ously for 4 h. The Et2O layer was separated. Drying,
evaporation and chromatography (EtOAc/hexane 1:1)
Incubation of these two peptides and the control peptide
58 showed, surprisingly, that the apparently conservative
replacement of Leu in previously published PSA-sensi-
tive peptides14 with Val led to the present SerLysLeuGln-
Val peptides being cleaved only very slowly by PSA and
at the N-terminal side of Gln, rather than at the previ-
ously observed C-terminal side14 of this residue. Further
design of PSA-cleavable peptide analogues will need to
reflect the apparently tight requirements in this region.
afforded 5 (500 mg, 6.5%) as a white solid: mp 154–
20
156 ꢁC; ½aꢀ +41ꢁ (c 3.2 · 10ꢂ3, CHCl3); IR mmax 3286,
D
1634, 1602, 1557, 1540 cmꢂ1; NMR dH 0.90 (6H, t,
J = 7.0 Hz, 2· Me), 1.41 (4H, sextet, J = 7.0 Hz, 2· Bu
3-CH2), 1.58 (4H, quintet, J = 7.0 Hz, 2· Bu 2-CH2),
3.01 (2H, dd, J = 14.8, 6.2 Hz, 2· 1-H), 3.07 (2H, dd,
J = 14.8, 6.2 Hz, 2· 1-H), 3.33 (4H, m, 2· Bu 1-CH2),
5.43 (2H, q, J = 6.2 Hz, 2· 2-H), 7.46–7.58 (8H, m,
2· Ar 3,4,5-H3+2· NH), 7.85 (4H, m, 2· Ar 2,6-H2),
7.96 (2H, t, J = 5.5 Hz, 2· NHBu); MS m/z 527.2696
(M+H) (C28H39N4O4S requires 527.2692); found: C,
62.30; H, 7.32; N, 10.37; C28H38N4O4S requires C,
63.85; H, 7.27; N, 10.63%. Further elution gave 6
5. Experimental
5.1. General
1H NMR spectra were recorded on Varian GX270 or
EX400 spectrometers of samples in CDCl3, unless other-
wise stated. IR spectra were recorded on a Perkin-Elmer
782 spectrometer as KBr discs, unless otherwise stated.
Mass spectra were obtained using fast atom bombard-
ment (FAB) ionisation in the positive ion mode. The
chromatographic stationary phase was silica gel. DCC
refers to N,N0-dicyclohexylcarbodiimide, THF refers to
tetrahydrofuran, DMF refers to dimethylformamide,
HOBt refers to 1-hydroxybenzotriazole, DMAP refers
to 4-dimethylaminopyridine, citric acid refers to a 5%
aqueous solution of 3-carboxy-3-hydroxypentanedioic
acid. THF was dried with Na. Solutions in organic sol-
vents were dried with MgSO4. Solvents were evaporated
under reduced pressure. The aqueous NaHCO3 and
brine were saturated. Experiments were conducted at
ambient temperature, unless otherwise stated. Melting
points were measured with a Thermo Galen Kofler
block and are uncorrected. All amino-acids and peptides
are of the L-configuration, except where noted. PSA was
purchased from Aldrich.
(310 mg, 4.0%) as a white solid: mp 178–180 ꢁC;
20
½aꢀ +0.0ꢁ (c 0.003, CHCl3); IR mmax 3287, 1633, 1602,
D
1560, 1532 cmꢂ1; NMR dH 0.93 (6H, t, J = 7.4 Hz,
2· Me), 1.40 (4H, sextet, J = 7.4 Hz, 2· Bu 3-CH2),
1.56 (4H, quintet, J = 7.4 Hz, 2· Bu 2-CH2), 3.14 (4H,
d, J = 6.2 Hz, 2· 2-H2), 3.33 (4H, m, 2· Bu 1-CH2),
4.90 (2H, q, J = 6.2 Hz, 2· 1-H), 7.33 (2H, t,
J = 5.5 Hz, 2· NHBu), 7.44 (6H, m, 2· Ph 3,5-
H2+2· NH), 7.53 (2H, m, 2· Ph 4-H), 7.81 (4H, m,
2· Ph 2,6-H2); MS m/z 527.2689 (M+H) (C14H31N4O2S2
requires 527.2692).
5.4. N,N-Diacetylcystine bis(naphth-2-yl)amide (10)
Cystine bis(naphth-2-yl)amide 9 (500 mg, 1.0 mmol) was
stirred with Ac2O (7 mL) and Et3N (206 mg, 2.0 mmol)
for 16 h. The evaporation residue was washed with H2O.
Drying afforded 10 (0.54 g, 92%) as a white solid: mp
250–252 ꢁC; IR mmax 3289, 1647, 1604, 1536,
1505 cmꢂ1; NMR ((CD3)2SO) dH 1.91 (6H, s, 2· Me),
3.03 (2H, dd, J = 13.3, 8.4 Hz, 2· b-H), 3.25 (2H, dd,
J = 13.3, 5.9 Hz, 2· b-H), 4.78 (2H, dt, J = 8.4, 5.9 Hz,
2· a-H), 7.40 (2H, dt, J = 1.2 Hz, 7.0 Hz) and 7.46
(2H, dt, J = 1.2, 7.0 Hz) (2· Ar 6,7-H2), 7.63 (2H, dd,
J = 8.6, 2.0 Hz, 2· Ar 3-H), 7.85 (6H, m, 2· Ar 4,5,8-
H3), 8.29 (2H, d, J = 2.0 Hz, 2· Ar 1-H), 8.45 (2H, d,
5.2. N,N-Dibenzoylcystine dihydrazide (8)
N,N0-Dibenzoylcystine dimethyl ester 726 (500 mg,
1.1 mmol) was stirred with N2H4ÆH2O (820 mg,
25 mmol) in MeOH (10 mL) for 16 h. The filtered solid
was washed with MeOH. Drying afforded 8 (300 mg,
60%) as a white solid: mp 205–207 ꢁC (lit.27 mp 206–
207 ꢁC); NMR dH 3.02 (2H, dd, J = 13.3, 9.8 Hz, 2· b-

G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
3481
J = 7.8 Hz, 2· NHAc), 10.41 (2H, s, 2· NH); MS m/z
575.1782 (M+H) (C30H31N4O4S2 requires 575.1787);
found: C, 60.70; H, 5.31; N, 9.18; C30H30N4O4S2 1.0
H2O requires C, 60.79; H, 5.44; N, 9.45%.
and chromatography (EtOAc ! EtOAc/MeOH 1:1)
afforded 19 (2.90 g, 71%) as a white solid: mp 145–
148 ꢁC; NMR dH 0.93 (3H, d, J = 6.6 Hz, Val-Me),
t
0.96 (3H, d, J = 6.6 Hz, Val-Me), 1.43 (9H, s, Bu),
2.04 (2H, m, Gln b-H2), 2.21 (1H, m, Val b-H), 2.42
(2H, t, J = 6.6 Hz, Gln c-H2), 3.73 (3H, s, OMe), 4.26
(1H, br q, J = 7.4 Hz, Gln a-H), 4.48 (1H, dd, J = 8.4,
5.1 Hz, Val a-H), 5.59 (1H, d, J = 7.4 Hz, Gln-NH),
5.84 (1H, s, CONH), 6.40 (1H, s, CONH), 7.59 (1H,
d, J = 8.4 Hz, Val NH); MS m/z 741 (2M+Na), 719
(2M+H), 382 (M+Na), 360.2142 (M+H) (C16H30N3O6
requires 360.2135); found C, 52.00; H, 7.82; N, 11.60;
C16H29N3O6 requires C, 52.14; H, 7.82; N, 11.60%.
5.5. N-Acetylcysteine naphth-2-ylamide (11)
Compound 10 (250 mg, 0.44 mmol) was boiled under re-
flux with propane-1,3-dithiol (480 mg, 4.4 mmol) and
Pri2NEt (142 mg, 1.1 mmol) in THF (10 mL) for 48 h.
The evaporation residue, in CH2Cl2, was washed with
cold aq H2SO4 (1 M, twice) and brine and was dried.
The evaporation residue was washed with hexane and
dried to afford 11 (190 mg, 75%) as a white solid: mp
204–207 ꢁC; IR mmax 3271, 1646, 1542, 1508 cmꢂ1
;
5.8. N-(N-(1,1-Dimethylethoxycarbonyl)glutaminyl)-
valine (20)
NMR dH 1.94 (1H, dd, J = 10.5, 7.0 Hz, SH), 2.15
(3H, s, Me), 2.81 (1H, ddd, J = 10.5, 7.4, 3.7 Hz, b-H),
3.24 (1H, ddd, J = 7.4, 7.0, 3.7 Hz, b-H), 4.80 (1H, dt,
J = 3.7, 7.0 Hz, a-H), 6.61 (1H, d, J = 6.6 Hz, NHAc),
7.47 (3H, m, Ar-H3), 7.79 (3H, m, Ar-H3), 8.19
(1H, d, J = 2.0 Hz, Ar 1-H), 8.74 (1H, br s, CONH);
MS m/z 288.0932 (M) (C15H16N2O2S requires 288.0932).
Compound 19 (890 mg, 2.5 mmol) was stirred with
NaOH (2.0 g, 50 mmol) in MeOH (30 mL) for 2 h.
H2O (10 mL) was added and MeOH was evaporated.
The solution was brought to neutrality with cold aq cit-
ric acid (10%) and extracted four times with EtOAc. The
aqueous layer was saturated with NaCl and extracted
further with EtOAc. Drying and evaporation of the
combined extracts afforded 20 (550 mg, 64%) as a white
solid: mp 86–89 ꢁC (lit.28 solid); NMR ((CD3)2SO) dH
0.87 (6H, d, J = 7.0 Hz, 2· Val-Me), 1.37 (9H, s, Boc),
1.66 (1H, m, Gln b-H), 1.83 (1H, m, Gln b-H), 2.10
(3H, m, Gln c-H2,Val b-H), 3.94 (1H, dt, J = 8.6,
5.5 Hz, Gln a-H), 4.15 (1H, dd, J = 8.6, 5.5 Hz, Val a-
H), 6.76 (1H, s, CONH), 6.94 (1H, d, J = 8.2 Hz, Gln
NH), 7.25 (1H, s, CONH), 7.75 (1H, d, J = 8.6 Hz,
Val NH); MS m/z 346.1975 (M+H) (C15H28N3O6
requires 346.1978).
5.6. N-Acetyl-S-(1-methoxy-1-methylethyl)cysteine
naphth-2-ylamide (13) and N-acetyl-S-(1-methylethe-
nyl)cysteine naphth-2-ylamide (14)
Compound 11 (50 mg, 0.17 mmol) was boiled under
reflux in 2,2-dimethoxypropane (0.7 mL), 4-methylben-
zenesulfonic acid hydrate (8.0 mg, 40 lmol) and CH2Cl2
(3 mL) for 3 h, after which CH2Cl2 (20 mL) and H2O
(10 mL) were added. The organic layer was extracted
and washed with H2O. Drying, evaporation and chro-
matography (EtOAc/hexane 9:1) afforded 14 (25 mg,
50%) as a colourless oil: NMR dH 2.03 (3H, s, Me),
2.10 (3H, s, COMe), 3.17 (1H, dd, J = 14.1, 7.4 Hz, b-
H), 3.30 (1H, dd, J = 14.1, 6.2 Hz, b-H), 4.87 (1H, dd,
J = 7.4, 6.2 Hz, a-H), 5.04 (1H, br s, @CH), 5.13 (1H,
s, @CH), 6.59 (1H, d, J = 7.0 Hz, AcNH), 7.45 (3H,
m, Ar 3,6,7-H3), 7.75 (3H, m, Ar 4,5,8-H3), 8.19 (1H,
d, J = 2.0 Hz, Ar 1-H), 8.85 (1H, br s, NH); MS m/z
329.1329 (M+H) (C19H25N2O3S requires 329.1324).
Further elution gave 13 (30 mg, 49%) as a white solid:
mp 137–138 ꢁC; NMR dH 1.56 (3H, s, Me), 1.58 (3H,
s, Me), 2.09 (3H, s, COMe), 3.03 (1H, dd, J = 13.7,
6.2 Hz, b-H), 3.10 (1H, dd, J = 13.7, 6.2 Hz, b-H), 3.67
(3H, s, OMe), 4.93 (1H, dt, J = 7.8, 6.2 Hz, a-H), 7.01
(1H, d, J = 7.8 Hz, AcNH), 7.39 (2H, m, Ar 6,7-H2),
7.46 (1H, dd, J = 9.0, 2.0 Hz, Ar 3-H), 8.20 (1H, d,
J = 1.6 Hz, Ar 1-H), 9.20 (1H, br s, NH); MS m/z
361.1589 (M+H) (C19H25N2O3S requires 361.1586).
5.9. N-(N-(1,1-Dimethylethoxycarbonyl)phenylalanyl)-
valine methyl ester (23)
BocPheOH (10.0 g, 37.7 mmol) was stirred with HOBt
(5.10 g, 37.7 mmol) and DCC (7.80 g, 37.7 mmol) in
CH2Cl2 (250 mL) at 0 ꢁC for 1 h. Compound 18
(6.30 g, 37.7 mmol) in CH2Cl2 (100 mL) was added in
the presence of Et3N (7.60 g, 75.4 mmol) and the mix-
ture was stirred for 48 h. The mixture was kept at 4 ꢁC
for 16 h and filtered through Celiteꢂ. The solution was
washed with 10% aq citric acid, aq NaHCO3 and
H2O. Drying, evaporation and recrystallisation
(EtOAc/hexane) afforded 23 (4.50 g, 33%) as a white
solid: mp 115–118 ꢁC (lit.29 mp 115–117 ꢁC); NMR dH
0.84 (3H, d, J = 7.0 Hz, Val-Me), 0.87 (3H, d,
J = 7.0 Hz, Val-Me), 1.42 (9H, s, Boc), 2.11 (1H, d sep-
tet, J = 5.0, 7.0 Hz, Val b-H), 3.07 (2H, d, J = 7.0 Hz,
Phe b-H2), 3.68 (3H, s, OMe), 4.34 (1H, br q,
J = 6.6 Hz, Phe a-H), 4.46 (1H, dd, J = 8.8, 5.1 Hz,
Val a-H), 4.99 (1H, br, Phe NH), 6.34 (1H, d,
J = 8.8 Hz, Val NH), 7.25 (5H, m, Ph-H5).
5.7. N-(N-(1,1-Dimethylethoxycarbonyl)glutami-
nyl)valine methyl ester (19)
BocGlnOH (2.78 g, 11.3 mmol) was stirred with HOBt
(1.68 g, 12.4 mmol) and DCC (3.49 g, 16.9 mmol) in
CH2Cl2 (150 mL) at 0 ꢁC for 1 h. Pri2NEt (2.92 g,
22.6 mmol) was added, followed by 18 (1.89 g,
11.3 mmol) in CH2Cl2 (50 mL) and the mixture was stir-
red for 24 h. The mixture was kept at 4 ꢁC for 16 h and
filtered (Celiteꢂ). The filtrate was washed with aq citric
acid (10%), aq NaHCO3 and H2O. Drying, evaporation
5.10. N-(N-(1,1-Dimethylethoxycarbonyl)phenylalanyl)-
valine (24)
Compound 23 (1.0 g, 2.6 mmol) was stirred with NaOH
(1.0 g, 26 mmol) in MeOH (15 mL) and H2O (15 mL)
for 1 h. The mixture was concentrated and EtOAc

3482
G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
(25 mL) was added. The mixture was brought to neu-
trality with cold 10% aq citric acid and extracted with
EtOAc. Drying and evaporation afforded 24 (850 mg,
88%) as a white solid: mp 77–78 ꢁC (lit.30 mp 77–
78 ꢁC); NMR dH 0.84 (3H, d, J = 7.0 Hz, Val-Me),
0.91 (3H, d, J = 7.0 Hz, Val-Me), 1.4 (9H, s, Boc),
2.17 (1H, d septet, J = 4.7, 6.7 Hz, Val b-H), 3.07 (2H,
m, Phe b-H2), 4.34 (1 H, m, Phe a-H), 4.46 (1H, dd,
J = 8.4, 4.7 Hz, Val a-H), 5.10 (1H, br, Phe NH), 6.55
(1H, d, J = 8.4 Hz, Val NH), 7.25 (5H, m, Ph-H5).
5.13. S-1-(1,1-Dimethylethoxycarbonyl)-5-oxopyrroli-
dine-2-carboxylic acid (31)
Compound 29 (15.0 g, 47 mmol) was stirred with 10%
Pd/C (450 mg) in MeOH (300 mL) under H2 for 6 h. Fil-
tration (Celiteꢂ) and evaporation afforded 31 (10.0 g,
93%) as a white solid: mp 65–67 ꢁC (lit.32 mp 77–
t
80 ꢁC); NMR ((CD3)2SO) dH 1.42 (9H, s, Bu), 1.90
(1H, m, Pyg b-H), 2.30–2.55 (3H, m, Pyg b,c-H3), 4.50
(1H, dd, J = 9.4, 3.5 Hz, Pyg a-H).
5.11. N-(N-(N-1,1-Dimethylethoxycarbonyl)phenylala-
nyl)valine N-(2-(4-nitrophenyl)ethyl)amide (26)
5.14. N-(S-1-(1,1-Dimethylethoxycarbonyl)-5-oxo-
pyrrolidine-2-carbonyl)valine phenylmethyl ester (32)
BocPheValOH 24 (200 mg, 0.55 mmol) was stirred with
pyridine (44 mg, 0.55 mmol) in dry CH2Cl2 (5 mL) and
added slowly to cyanuric fluoride (222 mg, 1.6 mmol)
in dry CH2Cl2 (5 mL) at ꢂ10 ꢁC. The mixture was stir-
red under N2 at ꢂ10 ꢁC for 2 h. The mixture was washed
thrice with ice-water. Drying and evaporation afforded
crude 25. This material, in CH2Cl2 (3 mL), was added
to 2-(4-nitrophenyl)ethylamine hydrochloride 3ÆHCl
(168 mg, 830 lmol), Et3N (172 mg, 1.7 mmol) and
DMAP (1 mg) in CH2Cl2 (3 mL) under N2. The mixture
was stirred for 16 h and the evaporation residue was
washed with EtOAc to afford 26 (100 mg, 36%) as a buff
solid: mp 202–203 ꢁC; NMR ((CD3)2SO) dH 0.76 (6H, d,
J = 6.7 Hz, 2· Val-Me), 1.28 (9H, s, Boc), 1.85 (1H, m,
Val b-H), 2.71 (1H, dd, J = 13.5, 10.6 Hz, Phe b-H),
2.85 (2H, t, J = 7.0 Hz, ArCH2), 2.91 (1H, dd,
J = 13.5, 4.1 Hz, Phe b-H), 3.34 (2H, m, NCH2), 4.07
(1H, dd, J = 9.1, 7.0 Hz, Val a-H), 4.17 (1H, ddd,
J = 10.3, 8.5, 4.1 Hz, Phe b-H), 7.03 (1H, d,
J = 8.5 Hz, Phe NH), 7.12–7.26 (5H, m, Ph-H5), 7.47
(2H, d, J = 8.8 Hz, Ar 2,6-H2), 7.64 (1H, d, J = 8.8 Hz,
Val NH), 8.06 (1H, t, J = 5.9 Hz, NH), 8.11 (2H, d,
J = 8.8 Hz, Ar 3,5-H2); MS m/z 513.2714 (M+H)
(C27H37N4O6 requires 513.2713), 457 (MꢂH2C@CMe2),
413 (MꢂBoc).
Compound 31 (10.0 g, 43.7 mmol) was stirred with
HOBt (6.50 g, 48.1 mmol) and DCC (9.92 g, 48.1 mmol)
in CH2Cl2 (200 mL) at 0 ꢁC for 1 h. ValOBn (16.60 g,
43.7 mmol) and Pri2NEt (11.30 g, 87.4 mmol) in CH2Cl2
(50 mL) were added and the mixture was stirred for 24 h.
The mixture was kept at 4 ꢁC for 16 h and filtered (Cel-
iteꢂ). The solution was washed with cold 5% aq citric
acid, aq NaHCO3 and H2O. Drying, evaporation and
chromatography (EtOAc) afforded 32 (13.0 g, 71%) as
a white solid: mp 96–98 ꢁC; NMR dH 0.87 (3H, d,
J = 7.0 Hz, Val-Me), 0.93 (3H, d, J = 7.0 Hz, Val-Me),
1.51 (9H, s, Boc), 2.20 (3H, m, Val b-H, Pyg b-H2),
2.46 (1H, ddd, J = 17.6, 8.5, 3.5 Hz, Pyg c-H), 2.73
(1H, dt, J = 17.6, 10.5 Hz, Pyg c-H), 4.57 (1H, dd,
J = 7.8, 3.1 Hz, Pyg a-H), 4.60 (1H, dd, J = 9.0, 4.7 Hz,
Val a-H), 5.14 (1H, d, J = 12.1 Hz) and 5.20 (1H, d,
J = 12.1 Hz) (CH2Ph), 6.46 (1H, d, J = 9.0 Hz, Val
NH), 7.35 (5H, m, Ph-H5); MS m/z 859 (2M+Na), 837
(2M+H), 737 (2M+H–Boc), 441 (M+Na), 419.2192
(M+H) (C22H31N2O6 requires 410.2182), 319 (MꢂBoc).
5.15. N-(S-1-(1,1-Dimethylethoxycarbonyl)-5-oxopyrr-
olidine-2-carbonyl)valine (33)
Compound 32 (7.50 g, 18 mmol) was stirred with 10%
Pd/C (400 mg) in MeOH (300 mL) under H2 for 16 h.
Filtration (Celiteꢂ) and evaporation afforded 33
(5.60 g, quant.) as a white solid: mp 152–155 ꢁC;
NMR ((CD3)2SO) dH 0.91 (3H, d, J = 7.0 Hz, Val-
Me), 0.92 (3H, d, J = 7.0 Hz, Val-Me), 1.39 (9H, s,
Boc), 1.80 (1H, m, Pyg b-H), 2.10 (1H, octet,
J = 7.0 Hz, Val b-H), 2.2–2.4 (3 H, m, Pyg b,c-H3),
4.16 (1H, dd, J = 8.2, 7.0 Hz, Val a-H), 4.68 (1H, dd,
J = 9.0, 2.3 Hz, Pyg a-H), 8.32 (1H, d, J = 8.6 Hz,
NH); MS m/z 679 (2M+Na), 657 (2M+H), 557
(2M+H–Boc), 351 (M+Na), 329.1721 (M+H)
(C15H25N2O6 requires 329.1713), 229 (MꢂBoc).
5.12. Phenylmethyl S-1-(1,1-dimethylethoxycarbonyl)-5-
oxopyrrolidine-2-carboxylate (29)
S-5-oxopyrrolidine-2-carboxylic
acid
27
(5.0 g,
39 mmol) was boiled under reflux with Et3N (3.92 g,
39 mmol) and benzyl chloride (5.4 g, 43 mmol) in THF
(50 mL) for 5 d. After cooling, H2O (50 mL) was added
and the THF was evaporated. The evaporation residue
was extracted thrice with CH2Cl2. The evaporation res-
idue was stirred with Et3N (3.92 g, 39 mmol), Boc2O
(16.9 g, 77.6 mmol) and DMAP (2.8 g, 39 mmol) in
CH2Cl2 (150 mL) at 0 ꢁC for 1 h and at 20 ꢁC for
16 h. The evaporation residue, in CH2Cl2, was washed
with cold 5% aq citric acid and brine. Drying and evap-
oration afforded 29 (9.0 g, 72%) as pale buff solid: mp
62–65 ꢁC (lit.31 mp 57–59 ꢁC); NMR dH 1.41 (9H, s,
But), 2.05 (1H, ddt, J = 12.8, 10.3, 3.3 Hz, Pyg b-H),
2.32 (1H, ddt, J = 12.8, 9.8, 9.4 Hz, Pyg b-H), 2.47
(1H, ddd, J = 17.6, 9.4, 3.5 Hz, Pyg c-H), 2.60 (1H,
ddd, J = 17.5, 10.3, 9.8 Hz, Pyg c-H), 4.62 (1H, dd,
J = 9.4, 3.3 Hz, Pyg a-H), 5.18 (1H, d, J = 12.1 Hz)
and 5.20 (1H, d, J = 12.1 Hz) (CH2Ph), 7.34 (5H, m,
Ph-H5).
5.16. N-(S-1-(1,1-Dimethylethoxycarbonyl)-5-oxopyrr-
olidine-2-carbonyl)valine pentafluorophenyl ester (34)
Compound 33 (200 mg, 610 lmol) was stirred with pen-
tafluorophenyl trifluoroacetate (188 mg, 671 lmol) and
pyridine (53 mg, 671 lmol) in dry DMF for 1 h. The
mixture was diluted with EtOAc (20 mL) and washed
with cold 5% aq citric acid and 5% aq NaHCO3. Drying
and evaporation afforded 34 (250 mg, 83%) as a highly
viscous colourless oil; NMR dH 1.05 (3H, d,
J = 7.0 Hz, Val-Me), 1.07 (3H, d, J = 6.6 Hz, Val-Me),

G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
3483
1.52 (9H, s, Boc), 2.26 (2H, m, Val b-H, Pyg b-H), 2.42
(1H, m, Pyg b-H), 2.50 (1H, m, Pyg c-H), 2.75 (1H, m,
Pyg c-H), 4.62 (1H, dd, J = 7.8, 3.1 Hz, Pyg a-H), 4.89
(1H, dd, J = 8.6, 5.1 Hz, Val a-H), 6.64 (1H, d,
J = 8.6 Hz, NH); NMR dF ꢂ161.2 (2F, dd, J = 20.9,
18.0 Hz, 30,50-F2), ꢂ156.4 (1F, t, J = 20.9 Hz, 40-F),
ꢂ151.6 (2 F, d, J = 18.0 Hz, 20,60-F2); MS m/z
495.1571 (M+H) (C21H24N2O6F5 requires 495.1555),
395 (MꢂBoc).
(3H, s, 2-Me), 1.92 (3H, s, 2-Me), 2.15 (2H, m, Val b-H,
Pyg b-H), 2.26 (1H, m, Pyg b-H), 2.50 (1H, ddd,
J = 17.2, 9.0, 2.7 Hz, Pyg c-H), 2.71 (1H, ddd,
J = 17.2, 10.5, 9.4 Hz, Pyg c-H), 3.46 (2H, m, 5-H2),
4.12 (1H, t, J = 8.6 Hz, Val a-H), 4.58 (1H, dd,
J = 9.0, 2.3 Hz, Pyg a-H), 6.14 (1H, dd, J = 4.3,
2.3 Hz, 4-H), 6.83 (1 H, d, J = 8.6 Hz, Val NH); NMR
dF ꢂ161.1 (2F, dd, J = 21.0, 18.4 Hz, 30,50-F2), ꢂ156.4
(1F, t, J = 21.0 Hz, 40-F), ꢂ151.4 (2F, d, J = 17.1 Hz,
20,60-F2); MS m/z 1298 (2M+Na), 1276 (2M+H),
5.17. S-N-(N-(S-1-(1,1-Dimethylethoxycarbonyl)-5-oxo-
pyrrolidine-2-carbonyl)valyl)-2,2-dimethyltetrahydro-
thiazole-4-carboxylic acid (35a) and S-N-(N-(S-1-(1,1-
dimethylethoxycarbonyl)-5-oxopyrrolidine-2-carbonyl)-D-
valyl)-2,2-dimethyltetrahydrothiazole-4-carboxylic acid
(35b)
660.1779
(M+Na)
(C27H32F5N3NaO7
requires
t
660.1795), 560 (M+Na–Boc), 538 (Mꢂ BuO).
5.19. S-3-(N-(S-1-(1,1-Dimethylethoxycarbonyl)-5-oxo-
pyrrolidine-2-carbonyl)valyl)-2,2-dimethyl-N-(2-(4-nitro-
phenyl)ethyl)tetrahydrothiazole-4-carboxamide (37)
Compound 34 (750 mg, 1.52 mmol) was stirred with
S-2,2-dimethyltetrahydrothiazole-4-carboxylic acid 21
(326 mg, 1.65 mmol) and Pri2NEt (640 mg, 4.95 mmol)
in dry DMF (100 mL) for 16 h. The evaporation residue,
in EtOAc, was washed with cold 5% aq citric acid and
brine. Drying, evaporation and chromatography
(EtOAc/AcOH 49:1) afforded 35a (300 mg, 42%) as a
white solid: mp 166–168 ꢁC; NMR dH 0.92 (3H, d,
J = 6.6 Hz, Val-Me), 0.95 (3H, d, J = 6.6 Hz, Val-Me),
1.50 (9H, s, Boc), 1.83 (3H, s, 2-Me), 1.88 (3H, s,
2-Me), 2.00–2.16 (2H, m, Val b-H, Pyg b-H), 2.25
(1H, m, Pyg b-H), 2.45 (1H, ddd, J = 17.7, 9.4, 2.7 Hz,
Pyg c-H), 2.69 (1H, dt, J = 17.7, 9.8 Hz, Pyg c-H),
3.26 (1H, dd, J = 12.1, 5.9 Hz, 5-H), 3.39 (1H, d,
J = 12.1 Hz, 5-H), 4.25 (1H, t, J = 9.4 Hz, Val a-H),
4.55 (1H, dd, J = 9.0, 2.0 Hz, Pyg a-H), 5.54 (1H, d,
J = 5.1 Hz, 4-H), 6.83 (1H, d, J = 9.4 Hz, Val-NH)
(Minor peaks were observed corresponding to a rot-
amer); MS m/z 472.2129 (M+H) (C21H34N3O7S requires
472.2117), 372 (MꢂBoc), 625 (M+mNBA+H), 742
(2· (MꢂBoc)). Further elution gave 35b (70 mg, 10%)
as a white solid: mp 149–151 ꢁC; NMR dH 0.93 (3H,
d, J = 6.6 Hz, Val-Me), 1.01 (3H, d, J = 6.6 Hz, Val-
Me), 1.50 (9H, s, Boc), 1.83 (3H, s, 2-Me), 1.87 (3H, s,
2-Me), 1.95 (2H, m, Val b-H, Pyg b-H), 2.45 (1H, m,
Pyg b-H), 2.48 (1H, ddd, J = 17.4, 9.4, 2.7 Hz, Pyg c-
H), 2.70 (1H, dt, J = 17.4, 9.8 Hz, Pyg c-H), 3.26 (1H,
dd, J = 11.7, 5.5 Hz, 5-H), 3.43 (1H, d, J = 11.7 Hz, 5-
H), 4.52 (1H, dd, J = 9.4, 2.7 Hz, Pyg a-H), 4.78 (1H,
dd, J = 9.4, 6.6 Hz, Val a-H), 5.02 (1H, d, J = 5.5 Hz,
4-H), 7.07 (1H, d, J = 9.4 Hz, Val-NH); MS m/z
471.2002 (MꢂH) (C21H32N3O7S requires 471.2039).
Compound 3: HCl (426 mg, 2.1 mmol) was stirred with
Et3N (425 mg, 4.2 mmol) and 36 (1.34 g, 2.1 mmol) in
CH2Cl2 (10 mL) for 5 h. Evaporation and chromatogra-
phy (EtOAc) afforded 37 (900 mg, 69%) as a pale buff
solid: mp 115–117 ꢁC; NMR dH 0.77 (3 H, d,
J = 6.6 Hz, Val-Me), 0.83 (3H, d, J = 6.6 Hz, Val-Me),
1.43 (9H, s, Boc), 1.70 (3H, s, 2-Me), 1.74 (3H, s, 2-
Me), 1.9–2.2 (3H, m, Val b-H, Pyg b-H2), 2.36 (1H,
ddd, J = 17.6, 9.4, 2.7 Hz, Pyg c-H), 2.57 (1H, m, Pyg
c-H), 2.90 (2H, t, J = 7.4 Hz, ArCH2), 2.93 (1H, d,
J = 12.5 Hz, 5-H), 3.28 (1H, dd, J = 12.5, 6.6 Hz, 5-H),
3.40 (1H, m, NHCH), 3.65 (1H, m, NHCH), 4.13 (1H,
t, J = 8.2 Hz, Val a-H), 4.46 (1H, dd, J = 9.0, 2.0 Hz,
Pyg a-H), 5.24 (1H, t, J = 6.6 Hz, 4-H), 6.39 (1H, d,
J = 8.2 Hz, Val NH), 6.50 (1H, t, J = 5.9 Hz, NHCH2),
7.31 (2H, d, J = 8.4 Hz, Ar 2,6-H2), 8.08 (2H, d,
J = 8.4 Hz, Ar 3,5-H2); MS m/z 520.2225 (MꢂBoc)
(C24H34N5O6S requires 520.2230).
5.20. S-3-(N-(N-(Dimethylethoxycarbonyl)glutami-
nyl)valyl)-2,2-dimethyl-N-(2-(4-nitrophenyl)ethyl)tetrahy-
drothiazole-4-carboxamide (38)
Compound 37 (100 mg, 162 lmol) was stirred in aq NH3
(35%, 1.0 mL) and THF (10 mL) for 18 h. Evaporation
afforded 38 (quant.) as a white solid: mp 95–97 ꢁC;
NMR dH 0.84 (3H, d, J = 6.6 Hz, Val-Me), 0.91 (3H,
d, J = 6.6 Hz, Val-Me), 1.42 (9H, s, Boc), 1.77 (3H, s,
2-Me), 1.79 (3H, s, 2-Me), 2.0 (3 H, m, Val b-H, Gln
b-H2), 2.25 (1H, m, Gln c-H), 2.35 (1H, m, Gln c-H),
2.95–3.00 (3H, m, 5-H, ArCH2), 3.37 (1H, dd,
J = 12.5, 6.2 Hz, 5-H), 3.45 (1H, m, NHCH), 3.75
(1H, m, NHCH), 4.11 (2H, m, Val a-H, Gln a-H),
5.35 (1H, d, J = 6.2 Hz, 4-H), 5.50 (1H, d, J = 7.4 Hz,
NH), 5.69 (1H, s, CONH), 6.21 (1H, s, CONH), 6.50
(1H, br t, NHCH2), 7.27 (1H, d, J = 8.2 Hz, NH), 7.38
(2H, d, J = 8.6 Hz, Ar 2,6-H2), 8.16 (2H, d, J = 8.6 Hz,
Ar 3,5-H2); MS m/z 637.3020 (M+H) (C29H45N6O8S re-
quires 637.3021), 537 (MꢂBoc).
5.18. Pentafluorophenyl S-N-(N-(S-1-(1,1-Dimethyleth-
oxycarbonyl)-5-oxopyrrolidine-2-carbonyl)valyl)-2,2-
dimethyltetrahydrothiazole-4-carboxylate (36)
Compound 35a (100 mg, 212 lmol) was stirred with
pentafluorophenyl trifluoroacetate (65 mg, 233 lmol)
and pyridine (18 mg, 233 lmol) in dry DMF (1 mL)
for 1 h. The mixture was diluted with EtOAc (20 mL)
and washed with cold 5% aq citric acid and 5% aq NaH-
CO3. Drying, evaporation and recrystallisation (EtOAc/
hexane) afforded 36 (125 mg, 93%) as a white solid: mp
150–152 ꢁC; NMR dH 0.92 (3H, d, J = 6.6 Hz, Val-Me),
0.96 (3H, d, J = 6.6 Hz, Val-Me), 1.54 (9H, s, Boc), 1.87
5.21. S-2,2-Dimethyl-3-(N-glutaminylvalyl)-N-(2-(4-
nitrophenyl)ethyl)tetrahydrothiazole-4-carboxamide
trifluoroacetate salt (39)
Compound 38 (300 mg, 472 lmol) was stirred in
CF3CO2H (900 lL) and CH2Cl2 (3.6 mL) for 1 h.

3484
G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
Evaporation and trituration (Et2O) afforded 39 (300 mg,
98%) as a white solid: mp 108–110 ꢁC; NMR (CD3OD) dH
0.89 (3H, d, J = 6.6 Hz, Val-Me), 0.91 (3H, d, J = 6.6 Hz,
Val-Me), 1.77 (3H, s, 2-Me), 1.88 (3H, s, 2-Me), 2.10 (3H,
m, Val b-H, Gln b-H2), 2.40 (2H, m, Gln c-H2), 2.99 (3H,
m, 5-H, ArCH2), 3.37 (1H, dd, J = 12.5, 6.2 Hz, 5-H), 3.51
(2H, m, NHCH2), 3.96 (1H, t, J = 6.6 Hz, Gln a-H), 3.98
(1H, d, J = 8.6 Hz, Val a-H), 5.38 (1H, d, J = 5.5 Hz, 4-
H), 7.48 (2H, d, J = 8.8 Hz, Ar 2,6-H2), 8.16 (2H, d,
J = 8.8 Hz, Ar 3,5-H2); MS m/z 537.2495 (M+H)
(C24H37N6O6S requires 537.2493).
5.24. Ne-(1,1-Dimethylethoxycarbonyl)-Na-(fluoren-9-
ylmethoxycarbonyl)lysine pentafluorophenyl ester (43)
Na-Fmoc-Ne-BocLysOH 42 (1.0 g, 2.1 mmol) was stir-
red with pentafluorophenol (432 mg, 2.3 mmol) and
DCC (483 g, 2.3 mmol) in EtOAc (5 mL) and THF
(5 mL) at 0 ꢁC under N2 for 4 h for 16 h. The mixture
was filtered (Celiteꢂ) and the precipitate was washed
with cold EtOAc. Evaporation afforded 43 (2.10 g,
70%) as a white solid: mp 102–104 ꢁC (lit.33 mp 99–
101 ꢁC); NMR dH 1.43 (9H, s, Boc), 1.50–2.85 (8H, m,
b,c,d,e-H8), 4.23 (1H, t, J = 7.0 Hz, CHCH2O), 4.41
(1H, dd, J = 10.5, 7.0 Hz, CHO), 4.49 (1H, dd,
J = 10.5, 7.0 Hz, CHO), 4.61 (1H, br, NH), 4.69 (1H,
q, J = 7.8 Hz, a-H), 5.57 (1H, d, J = 7.0 Hz, NH), 7.29
(2H, t, J = 7.4 Hz, Ar-H2), 7.38 (2H, t, J = 7.4 Hz, Ar-
H2), 7.58 (2H, d, J = 7.4 Hz, Ar-H2), 7.74 (2H, d,
J = 7.4 Hz, Ar-H2); NMR dF ꢂ161.7 (2F, t,
J = 20.7 Hz, 30,50-F2), ꢂ157.1 (1F, t, J = 20.7 Hz, 40-
F), ꢂ152.2 (2F, d, J = 18.4 Hz, 20,6F2).
5.22. S-2,2-Dimethyl-3-(N-(N-(N-(dimethylethoxycar-
bonyl)leucyl)glutaminyl)valyl)-N-(2-(4- nitrophenyl)ethyl)-
tetrahydrothiazole-4-carboxamide (40)
BocLeuOSu (157 mg, 477 lmol) in dry THF (2.0 mL)
was added to 39 (310 mg, 477 lmol), Et3N (97 mg,
954 lmol) and DMAP (1 mg) in dry DMF (1.5 mL) at
0 ꢁC and the mixture was stirred for 30 min. The mixture
was warmed to 20 ꢁC and stirred for 24 h. The evapora-
tion residue, in EtOAc, was washed with cold 5% aq
citric acid, H2O and brine. Drying, evaporation and
recrystallisation (EtOAc/hexane) afforded 40 (330 mg,
92%) as white solid: mp 109–111 ꢁC; NMR (CD3OD)
dH 0.85 (3H, d, J = 6.6 Hz, Val-Me), 0.86 (3H, d,
J = 6.6 Hz, Val-Me), 0.93 (3H, d, J = 6.6 Hz, Leu-Me),
0.95 (3H, d, J = 6.6 Hz, Leu-Me), 1.45 (9H, s, Boc), 1.52
(2H, t, J = 7.0 Hz, Leu b-H2), 1.79 (3H, s, 2-Me), 1.88
(3H, s, 2-Me), 1.70–2.10 (4H, m, Val b-H, Leu c-H,
Glnb-H2), 2.30 (2H, m, Gln c-H2), 2.94–3.04 (3H, m, 5-H,
ArCH2), 3.42 (1H, dd, J = 12.1, 5.9 Hz, 5-H), 3.55 (2H,
br q, J = 5.5 Hz, NHCH2), 3.95 (1H, t, J = 9.0 Hz, Val
a-H), 4.04 (1H, t, J = 7.4 Hz, Leu a-H), 4.30 (1H, br q,
J = 6.6 Hz, Gln a-H), 5.21 (1H, d, J = 5.9 Hz, 4-H), 7.50
(2H, d, J = 8.6 Hz, Ar 2,6-H2), 8.07 (1H, t, J = 5.5 Hz,
NHCH2), 8.16 (2H, d, J = 8.6 Hz, Ar 3,5-H2), 8.18 (2H,
m, 2· NH); MS m/z 772 (M+Na), 750.3860 (M+H)
(C35H56N7O9S requires 750.3901), 650 (MꢂBoc).
5.25. S-2,2-Dimethyl-3-(N-(N-(N-(Ne-(1,1-dimethyleth-
oxycarbonyl)-Na-(fluoren-9-ylmethoxycarbonyl)lysyl)-
leucyl)glutaminyl)valyl)-N-(2-(4-nitrophenyl)ethyl)tetra-
hydrothiazole-4-carboxamide (44)
Compound 43 (290 mg, 380 lmol) in dry THF (1.5 mL)
was stirred with 41 (241 mg, 380 lmol), Et3N (77 mg,
760 lmol) and DMAP (1 mg) in dry DMF (1.5 mL) at
0 ꢁC for 30 min. The mixture was warmed to 20 ꢁC and
stirred for 16 h. The evaporation residue, in EtOAc, was
washed with cold 5% aqcitric acid, H2O and brine. Drying
and recrystallisation (EtOAc/hexane) afforded 44
(395 mg, 95%) as a white solid: mp 72–75 ꢁC; NMR dH
0.75–0.85 (12H, m, 2· Val-Me, 2· Leu-Me), 1.20–1.40
(6H, m, Lys b,c,d-H6), 1.43 (9H, s, Boc), 1.50–1.65 (3H,
m, Leu b,c-H3), 1.73 (3H, s, 2-Me), 1.83 (3H, s, 2-Me),
1.90–2.10 (3H, m, Val b-H, Gln b-H2), 2.30 (2H, m, Gln
c-H2), 2.78 (2H, t, J = 7.4 Hz, Lys e-H2), 2.89–3.03 (4H,
m, 5-H2, ArCH2), 3.35 (1H, m, NHCHCH2), 3.65 (1H,
m, NHCHCH2), 4.04 (1H, t, J = 9.0 Hz, CHCH2O),
4.15–4.50 (6H, m, CH2O, Val a-H, Gln a-H, Leu a-H,
Lys a-H), 5.11 (1H, m, 4-H), 7.24–7.55 (8H, m, Ar-H8),
7.72 (2H, d, J = 7.4 Hz, Ar 2,6-H2), 8.10 (2H, d,
J = 7.4 Hz, Ar 3,5-H2); MS m/z 1100.5544 (M+H)
(C56H78N9O12S requires 1100.5491), 1000 (MꢂBoc).
5.23. S-2,2-Dimethyl-3-(N-(N-leucylglutaminyl)valyl)-N-
(2-(4-nitrophenyl)ethyl)tetrahydrothiazole-4-carboxamide
(41)
Compound 40 (300 mg, 401 lmol) was stirred in
CF3CO2H (0.9 mL) and CH2Cl2 (3.6 mL) for 30 min.
Evaporation and trituration (Et2O) afforded 41
(300 mg, 98%) as a white solid: mp 121–123 ꢁC; NMR
(CD3OD) dH 0.76 (3H, d, J = 7.0 Hz, Val-Me), 0.77
(3H, d, J = 6.6 Hz, Val-Me), 1.00 (3H, d, J = 5.5 Hz,
Leu-Me), 1.01 (3H, d, J = 5.5 Hz, Leu-Me), 1.54–1.74
(3H, m, Leu b-H2, Leu c-H), 1.70 (3H, s, 2-Me), 1.79
(3H, s, 2-Me), 1.82–2.04 (3H, m, Val b-H, Gln b-H2),
2.20 (2H, m, Gln c-H2), 2.89 (2H, t, J = 6.6 Hz, ArCH2),
2.91 (1H, d, J = 12.1 Hz, 5-H), 3.30 (1H, dd, J = 12.1,
5.9 Hz, 5-H), 3.50 (2H, m, NHCH2), 3.80 (1H, br t,
J = 8.2 Hz, Leu a-H), 3.87 (1H, d, J = 9.4 Hz, Val a-
H), 4.31 (1H, t, J = 7.8 Hz, Gln a-H), 5.20 (1H, d,
J = 5.9 Hz, 4-H), 7.40 (2H, d, J = 9.0 Hz, Ar 2,6-H2),
7.97 (1H, t, J = 5.5 Hz, NHCH2), 8.06 (2H, d,
J = 9.0 Hz, Ar 3,5-H2), 8.26 (1H, d, J = 9.0 Hz, NH);
MS m/z 650.3343 (M+H) (C30H48N7O7S requires
650.3336), 723 (M+Na).
5.26. S-2,2-Dimethyl-3-(N-(N-(N-(Ne-(1,1-dimethyleth-
oxycarbonyl)lysyl)leucyl)glutaminyl)valyl)-N-(2-(4-nitro-
phenyl)ethyl)tetrahydrothiazole-4-carboxamide (45)
Compound 44 (250 mg, 227 lmol) was stirred in CH2Cl2
(4.5 mL) and diethylamine (500 lL) for 30 min. The
evaporation residue was washed with Et2O and dried
to afford 45 (180 mg, 90%) as a white solid: mp 100–
103 ꢁC; NMR (CD3OD) dH 0.85 (3H, d, J = 6.6 Hz,
Val-Me), 0.86 (3H, d, J = 6.6 Hz, Val-Me), 0.92 (3H,
d, J = 6.6 Hz, Leu-Me), 0.96 (3H, d, J = 6.6 Hz, Leu-
Me), 1.40–1.65 (18H, m, Boc, Lys b,c,d-H6, Leu b,c-
H3), 1.79 (3H, s, 2-Me), 1.88 (3H, s, 2-Me), 1.90–2.12
(3H, m, Val b-H, Gln b-H2), 2.30 (2H, m, Gln c-H2),
2.96–3.04 (6H, m, Lys e-H2, 5-H2, ArCH2), 3.40 (1H,
m, NHCHCH2), 3.55 (1H, m, NHCHCH2), 3.92–4.42

G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
3485
(4H, m, Val a-H, Gln a-H, Leu a-H, Lys a-H), 5.24 (1H,
5.29. S-2,2-Dimethyl-3-(N-(N-(N-(Ne-(1,1-dimethyleth-
d, J = 5.4 Hz, 4-H), 7.50 (2H, d, J = 8.6 Hz, Ar 2,6-H2),
8.15 (2H, d, J = 8.6 Hz, Ar 3,5-H2); MS m/z 878.4831
(M+H) (C41H68N9O10S requires 878.4810), 778
(MꢂBoc).
oxycarbonyl)-Na-(N-(1,1-dimethylethyl)-N-(fluoren-9-
ylmethoxycarbonyl)serinyl)lysyl)leucyl)glutaminyl)valyl)-
N-(2-(4-nitrophenyl)ethyl)tetrahydrothiazole-4-carbox-
amide (48)
5.27. S-2,2-Dimethyl-3-(N-(N-(N-(Ne-(1,1-dimethyleth-
oxycarbonyl)-Na-(O-(1,1-dimethylethyl)-N-(phenylmeth-
oxycarbonyl)serinyl)lysyl)leucyl)glutaminyl)valyl)-N-(2-
(4-nitrophenyl)ethyl)tetrahydrothiazole-4-carboxamide
(46)
FmocSer(But)OSu (44 mg, 91 lmol) in dry THF (500 lL)
was stirred with 45 (80 mg, 91 lmol), Et3N (18.4 mg,
182 lmol) and DMAP (1 mg) in dry DMF (500 lL) at
0 ꢁC for 30 min and at 20 ꢁC for 16 h. The evaporation
residue, in EtOAc, was washed with cold 5% aq citric acid,
H2O and brine. Drying and recrystallisation (EtOAc/hex-
ane) afforded 48 (70 mg, 62%) as a white solid: mp 184–
186 ꢁC; NMR (CD3OD) dH 0.85 (3H, d, J = 6.6 Hz,
Val-Me), 0.86 (3H, d, J = 6.6 Hz, Val-Me), 0.90 (3H, d,
J = 6.2 Hz, Leu-Me), 0.95 (3H, d, J = 6.2 Hz, Leu-Me),
1.18 (9H, s, SerOBut), 1.43 (9H, s, Boc), 1.45–1.75 (9H,
m, Lys b,c,d-H6, Leu b,c-H3), 1.79 (3H, s, 2-Me), 1.88
(3H, s, 2-Me), 1.90–2.15 (3H, m, Val b-H, Gln b-H2),
2.30 (2H, m, Gln c-H2), 2.96–3.02 (4H, m, ArCH2, Ser
b-H2), 3.15–3.25 (2H, m, 5-H2), 3.55 (2H, m, Lys e-H2),
3.62 (2H, m, NHCH2), 3.94 (1H, d, J = 9.4 Hz,
CHCH2O), 4.21–4.35 (5H, m, Gln a-H, Leu a-H, Lys a-
H, Ser a-H, fluorene 9-H), 5.20 (1H, d, J = 5.1 Hz, 4-H),
7.30 (2H, t, J = 7.4 Hz, Ar-H2), 7.39 (2H, t, J = 7.4 Hz,
Ar-H2), 7.50 (2H, d, J = 8.6 Hz, Ar 2,6-H2), 7.66 (2H,
J = 7.4 Hz, Ar-H2), 7.80 (2H, J = 7.4 Hz, Ar-H2),8.16
(2H, d, J = 8.6 Hz, Ar 3,5-H2); MS m/z 1243.6437
(M+H) (C63H91N10O14S requires 1243.6477).
CbzSer(But)OSu (13.3 mg, 34 lmol) in dry THF (700 lL)
was stirred with 45 (30 mg, 34 lmol), Et3N (6.9 mg,
68 lmol) and DMAP (1 mg) in dry DMF (300 lL) at
0 ꢁC for 30 min and at 20 ꢁC for 16 h. The evaporation
residue, in EtOAc, was washed with cold 5% aq citric acid,
H2O and brine. Drying and recrystallisation (EtOAc/hex-
ane) afforded 46 (20 mg, 51%) as a white solid: mp 128–
130 ꢁC; NMR (CD3OD) dH 0.84 (3H, d, J = 6.6 Hz,
Val-Me), 0.86 (3H, d, J = 6.6 Hz, Val-Me), 0.89 (3H, d,
J = 6.2 Hz, Leu-Me), 0.90 (3H, d, J = 6.2 Hz, Leu-Me),
1.20 (9H, s, SerOBut), 1.42 (9H, s, Boc), 1.45–1.75 (9H,
m, Lys b,c,d-H6, Leu b,c-H3), 1.79 (3H, s, 2-Me), 1.88
(3H, s, 2-Me), 1.90–2.15 (3H, m, Val b-H, Gln b-H2),
2.30 (2H, m, Gln c-H2), 2.96–3.04 (4H, m, Lys e-H2,
ArCH2), 3.42 (1H, dd, J = 12.5, 5.9 Hz, 5-H), 3.55 (3H,
m, 5-H, NHCH2), 3.76 (1H, m, Ser b-H), 3.85 (1H, m,
Ser b-H), 3.95 (1H, d, J = 9.4 Hz, Val a-H), 4.15–4.44
(4H, m, Gln a-H, Leu a-H, Lys a-H, Ser a-H), 5.09 (1H,
d, J = 12.5 Hz) and 5.14 (1H, d, J = 12.5 Hz) (CH2Ph),
5.21 (1H, d, J = 5.9 Hz, 4-H), 7.36 (5H, m, Ph-H5), 7.51
(2H, d, J = 8.8 Hz, Ar 2,6-H2), 8.06 (1H, br t, J = 7 Hz,
NH), 8.16 (2H, d, J = 8.8 Hz, Ar 3,5-H2); MS m/z
1155.6162 (M+H) (C56H87N10O14S requires 1155.6124),
1055 (MꢂBoc), 846 (Mꢂ DmtNHCH2CH2PhNO2).
5.30. S-2,2-Dimethyl-3-(N-(N-(N-(Ne-(1,1-dimethyleth-
oxycarbonyl)-Na-(N-(1,1-dimethylethyl)serinyl)lysyl)leu-
cyl)glutaminyl)valyl)-N-(2-(4-nitrophenyl)ethyl)tetrahy-
drothiazole-4-carboxamide (49)
Compound 48 (70 mg, 56 lmol) was stirred in CH2Cl2
(1.35 mL) and Et2NH (150 lL) for 30 min. The evapora-
tion residue was washed with Et2O and dried to afford
49 (50 mg, 88%) as a white solid: mp 94–96 ꢁC; NMR
(CD3OD) dH 0.85 (3H, d, J = 6.6 Hz, Val-Me), 0.86
(3H, d, J = 6.6 Hz, Val-Me), 0.92 (3H, d, J = 6.2 Hz,
Leu-Me), 0.96 (3H, d, J = 6.2 Hz, Leu-Me), 1.31 (9H,
s, SerOBut), 1.44 (9H, s, Boc), 1.45–1.75 (9H, m, Lys
b,c,d-H6, Leu b,c-H3), 1.78 (3H, s, 2-Me), 1.88 (3H, s,
2-Me), 1.90–2.15 (3H, m, Val b-H, Gln b-H2), 2.30
(2H, t, J = 7.0 Hz, Gln c-H2), 2.96–3.05 (5H, m, 5-H,
ArCH2, Ser b-H2), 3.42 (1H, dd, J = 12.1, 5.3 Hz, 5-
H), 3.55 (2H, m, Lys e-H2), 3.58–3.55 (2H, m, NHCH2),
3.95 (1H, d, J = 9.0 Hz, Val a-H), 4.20–4.40 (4H, m, Gln
a-H, Leu a-H, Lys a-H, Ser a-H), 5.21 (1H, d,
J = 5.3 Hz, 4-H), 7.51 (2H, d, J = 8.6 Hz, Ar 2,6-H2),
8.16 (2H, d, J = 8.6 Hz, Ar 3,5-H2); MS m/z 1021.5756
(M+H) (C48H81N10O12S requires 1021.5762).
5.28. S-2,2-Dimethyl-3-(N-(N-(N-(N-(N-(phenylmeth-
oxycarbonyl)serinyl)lysyl)leucyl)glutaminyl)valyl)-N-(2-
(4-nitrophenyl)ethyl)tetrahydrothiazole-4-carboxamide
trifluoroacetate salt (47)
Compound 46 (5.0 mg, 4.3 lmol) was stirred in
CF3CO2H (100 lL) and CH2Cl2 (400 lL) for 2 h. Evap-
oration and trituration (Et2O) afforded 47 (4.7 mg, 98%)
as a white solid: mp 107–109 ꢁC; NMR (CD3OD) dH
0.84 (3H, d, J = 6.6 Hz, Val-Me), 0.85 (3H, d,
J = 6.6 Hz, Val-Me), 0.90 (3H, d, J = 6.2 Hz, Leu-Me),
0.95 (3H, d, J = 6.2 Hz, Leu-Me), 1.45–1.70 (9H, m,
Lys b,c,d-H6, Leu b,c-H3), 1.78 (3H, s, 2-Me), 1.88
(3H, s, 2-Me), 1.90–2.15 (3H, m, Val b-H, Gln b-H2),
2.30 (2H, t, J = 7.4 Hz, Gln c-H2), 2.39 (2H, t,
J = 7.0 Hz, ArCH2), 2.98 (2H, t, J = 6.5 Hz, Lys e-H2),
3.03 (1H, J = 12.1 Hz, 5-H), 3.41 (1H, dd, J = 12.1,
5.5 Hz, 5-H), 3.55 (2H, m, NHCH2), 3.75 (1H, m, Ser
b-H), 3.85 (1H, m, Ser b-H), 3.95 (1H, d, J = 9.4 Hz,
Val a-H), 4.20–4.40 (4H, m, Gln a-H, Leu a-H, Lys a-
H, Ser a-H), 5.07 (1H, d, J = 12.5 Hz) and 5.12 (1H,
d, J = 12.5 Hz) (CH2Ph), 5.21 (1H, d, J = 5.5 Hz, 4-H),
7.36 (5H, m, Ph-H5), 7.51 (2H, d, J = 8.8 Hz, Ar 2,6-
H2), 8.06 (1H, br t, J = 7 Hz, NH), 8.16 (2H, d,
J = 8.8 Hz, Ar 3,5-H2); MS m/z 999.4964 (M+H)
(C57H71N10O12S requires 999.4974).
5.31. S-2,2-Dimethyl-3-(N-(N-(N-(Ne-(1,1-dimethyleth-
oxycarbonyl)-Na-(N-(1,1-dimethylethyl)-N-(N-(1,1-
dimethylethyl)-N-(phenylmethoxycarbonyl)serinyl)seri-
nyl)lysyl)leucyl)glutaminyl)valyl)-N-(2-(4-nitro-
phenyl)ethyl)tetrahydrothiazole-4-carboxamide (50)
CbzSer(But)OSu (9.8 mg, 25 lmol) in dry THF (700 lL)
was added to compound 49 (25 mg, 25 lmol), Et3N
(5.1 mg, 50 lmol) and DMAP (1 mg) in dry DMF

3486
G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
(300 lL) at 0 ꢁC. The mixture was stirred for 30 min at
0 ꢁC and for 16 h at 20 ꢁC. The evaporation residue, in
EtOAc, was washed with cold 5% aq citric acid, H2O
and brine. Drying and evaporation afforded 50 (20 mg,
62%) as a white solid: mp 202–205 ꢁC; NMR (CD3OD)
dH 0.86 (3H, d, J = 6.6 Hz, Val-Me), 0.89 (3H, d,
J = 6.6 Hz, Val-Me), 0.91 (3H, d, J = 5.9 Hz, Leu-Me),
0.95 (3H, d, J = 5.9 Hz, Leu-Me), 1.20 (18H, s, 2· SerO-
But), 1.42 (9H, s, Boc), 1.45–1.75 (9H, m, Lys b,c,d-H6,
Leu b,c-H3), 1.80 (3H, s, 2-Me), 1.88 (3H, s, 2-Me),
1.90–2.15 (3H, m, Val b-H, Gln b-H2), 2.30 (2H, m,
Gln c-H2), 2.96–3.04 (4H, m, Lys e-H2, ArCH2), 3.45–
3.8 (8H, m, 5-H2, NHCH2, 2· Ser b-H2), 3.95 (1H, d,
J = 9.4 Hz, Val a-H), 4.15–4.80 (6H, m, Gln a-H, Leu
a-H, Lys a-H, 2· Ser a-H), 5.09 (2H, m, CH2Ph), 5.21
(1H, d, J = 5.5 Hz, 4-H), 7.35 (5H, m, Ph-H5), 7.51
(2H, d, J = 8.8 Hz, Ar 2,6-H2), 8.16 (2H, d, J = 8.8 Hz,
(5%) and brine. Drying, evaporation and chromatogra-
phy (EtOAc ! EtOAc/MeOH 1:1) afforded 53 (900 mg,
54%) as a white solid: mp 135–139 ꢁC; NMR dH 0.83–
0.87 (12H, m, 2· Val-Me, 2· Leu-Me), 1.34 (9H, s,
Boc), 1.41 (1H, m, Leu b-H), 1.57 (1H, m, Leu c-H),
1.70 (2H, m, Gln b-H, Leu b-H), 1.85 (1H, m, Gln b-
H), 2.03 (1H, m, Val b-H), 2.08 (2H, m, Gln c-H2),
3.62 (3H, s, OMe), 3.94 (1H, dt, J = 8.2, 6.6 Hz, Leu
a-H), 4.14 (1H, dd, J = 7.8, 5.9 Hz, Val a-H), 4.34
(1H, dt, J = 7.8, 5.5 Hz, Gln a-H), 6.77 (1H, s, CONH),
6.95 (1H, d, J = 8.2 Hz, Leu-NH), 7.25 (1H, s, CONH),
7.82 (1H, d, J = 7.8 Hz, Gln NH), 8.12 (1H, d,
J = 7.8 Hz, Val NH); MS m/z 495.2793 (M+Na)
(C22H40N4O7 requires 495.2795), 395 (M+Na–Boc).
5.35. N-(N-Leucylglutaminyl)valine methyl ester
trifluoroacetate salt (54)
Ar
3,5-H2);
MS
m/z
1199.6588
(MꢂBoc)
(13C12C57H92N11O14S requires 1199.6579), 1297 (MꢂH).
Compound 53 (650 mg, 1.4 mmol) was stirred in
CF3CO2H (2 mL) and CH2Cl2 (2 mL) for 2 h. Evapora-
tion afforded 54 (quant.) as a highly hygroscopic colour-
less viscous oil: NMR ((CD3)2SO) dH 0.85–0.87 (12H,
m, 2· Val-Me, 2· Leu-Me), 1.26 (1H, m, Leu b-H), 1.62
(1H, m, Leu c-H), 1.73 (2H, m, Gln b-H, Leu b-H), 1.85
(1H, m, Gln b-H), 2.04 (1H, m, Val b-H), 2.15 (2H, m,
Gln c-H2), 3.62 (3H, s, OMe), 3.80 (1H, m, Leu a-H),
4.16 (1H, dd, J = 8.2, 6.2 Hz, Val a-H), 4.42 (1H, dt,
J = 7.8, 5.5 Hz Gln a-H), 6.82 (1H, s, CONH), 7.31
(1H, s, CONH), 8.14 (3H, br, N+H3), 8.28 (1H, d,
J = 8.2 Hz, Val NH), 8.66 (1H, d, J = 7.8 Hz, Gln NH);
MS m/z 525 (M+mNBA), 395 (M+Na), 373.2443
(M+H) (C17H33N4O5 requires 373.2451).
1
5.32. S-2,2-Dimethyl-3-(N-(N-(N-(N-(N-(N-(phenylmeth-
oxycarbonyl)serinyl)serinyl)lysyl)leucyl)glutaminyl)valyl)-
N-(2-(4-nitrophenyl)ethyl)tetrahydrothiazole-4-carbox-
amide trifluoroacetate (51)
Compound 50 (5.0 mg, 3.9 lmol) was stirred in
CF3CO2H (100 lL) and CH2Cl2 (400 lL) for 2 h. Evapo-
ration afforded 51 (quant.) as a highly hygroscopic gum-
my solid: NMR (CD3OD) dH 0.86 (3H, d, J = 6.6 Hz,
Val-Me), 0.89 (3H, d, J = 6.6 Hz, Val-Me), 0.91 (3H, d,
J = 5.9 Hz, Leu-Me), 0.95 (3H, d, J = 5.9 Hz, Leu-Me),
1.45–1.75 (9H, m, Lys b,c,d-H6, Leu b,c-H3), 1.80 (3H,
s, 2-Me), 1.88 (3H, s, 2-Me), 1.90–2.15 (3H, m, Val b-H,
Gln b-H2), 2.30 (2H, m, Gln c-H2), 2.96–3.04 (4H, m,
Lys e-H2, ArCH2), 3.45–3.8 (8H, m, 5-H2, NHCH2,
2· Ser b-H2), 3.95 (1H, d, J = 9.4 Hz, Val a-H), 4.00–
4.80 (6H, m, Gln a-H, Leu a-H, Lys a-H, 2· Ser a-H),
5.10 (2H, m, CH2Ph), 5.21 (1H, d, J = 5.5 Hz, 4-H),
7.35 (5H, m, Ph-H5), 7.51 (2H, d, J = 8.8 Hz, Ar 2,6-
H2), 8.16 (2H, d, J = 8.8 Hz, Ar 3,5-H2).
5.36. N-(N-(N-(Ne-(1,1-Dimethylethoxycarbonyl)-Na-
(fluoren-9-ylmethoxycarbonyl)lysyl)leucyl)glutami-
nyl)valine methyl ester (55)
Compound 43 (700 mg, 1.1 mmol) in dry THF (5 mL)
was stirred with 54 (826 mg, 1.7 mmol), Et3N (344 mg,
3.4 mmol) and DMAP (1 mg) in dry DMF (3 mL) at
0 ꢁC for 30 min and at 20 ꢁC for 16 h. The evaporation
residue was washed with EtOAc, cold aq citric acid (5%)
and H2O. Drying and evaporation afforded 55 (600 mg,
5.33. N-Glutaminylvaline methyl ester trifluoroacetate
salt (52)
68%) as
a
white solid: mp 200–202 ꢁC; NMR
Compound 19 (1.00 g, 2.8 mmol) was stirred in
CF3CO2H (3 mL) and CH2Cl2 (3 mL) for 45 min. Evapo-
ration afforded crude 52 (quant.) as a highly hygroscopic
yellow viscous oil: NMR ((CD3)2SO) dH 0.85 (6H, d,
J = 7.0 Hz, 2· Val-Me), 1.88 (2H, br q, J = 5.7 Hz, Gln
b-H2), 2.00 (1H, m, Val b-H), 2.20 (2H, t, J = 7.4 Hz,
Gln c-H2), 3.59 (3H, s, OMe), 3.87 (1H, br q,
J = 5.7 Hz, Gln a-H), 4.16 (1H, dd, J = 7.8, 5.9 Hz, Val
a-H), 6.93 (1H, s, CONH), 7.42 (1H, s, CONH), 8.14
(3H, s, N+H3), 8.68 (1H, d, J = 7.8 Hz, Val NH).
((CD3)2SO) dH 0.80–0.86 (12H, m, 2· Val-Me, 2· Leu-
Me), 1.35 (9 H, s, Boc), 1.44–1.52 (6H, m, Lys b,c,d-
H6), 1.54 (1H, m, Gln b-H), 1.57 (1H, m, Gln b-H),
2.05 (1H, m, Val b-H), 2.22 (2H, m, Gln c-H2), 2.88
(2H, m, Lys e-H2), 3.61 (3H, s, OMe), 3.94–4.32 (7H,
m, Leu a-H, Gln a-H, Val a-H, Lys a-H, fluorene 9-
H, CH2O), 6.77 (2H, m, CONH, NH), 7.23 (1H, s,
CONH), 7.30 (2H, t, J = 7.4 Hz, Ar-H2), 7.39 (2H, t,
J = 7.4 Hz, Ar-H2), 7.46 (1H, d, J = 8.2 Hz, NH), 7.69
(2H, t, J = 7.4 Hz Ar-H2), 7.86 (2H, d, J = 7.4 Hz, Ar-
H2), 7.89 (1H, d, J = 8.2 Hz, NH), 7.94 (1H, d,
J = 7.4 Hz, NH), 8.04 (1H, d, J = 8.2 Hz, NH); MS
m/z 823.4597 (M+H) (C43H63N6O10 requires 823.4606).
5.34. N-(N-(N-(1,1-Dimethylethoxycarbonyl)leucyl)glu-
taminyl)valine methyl ester (53)
BocLeuOSu (1.14 g, 3.5 mmol) in dry THF (5 mL) was
stirred with 52 (1.29 g, 3.5 mmol), Et3N (708 mg,
7 mmol) and DMAP (1 mg) in dry DMF (5 mL) at
0 ꢁC for 30 min and at 20 ꢁC for 24 h. The evaporation
residue, in EtOAc, was washed with cold aq citric acid
5.37. N-(N-(N-(Ne-(1,1-Dimethylethoxycarbonyl)lysyl)-
leucyl)glutaminyl)valine methyl ester (56)
Compound 55 (52 mg, 64 lmol) was stirred in DMF
(900 lL) and Et2NH (100 lL) for 30 min. The evaporation

G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
3487
residue was washed with Et2O and dried to afford 56
(35 mg, 91%) as a white solid: mp 162-165 ꢁC; NMR
(CD3OD) dH 0.94–0.98 (12H, m, 2· Val-Me, 2· Leu-
Me), 1.38–1.62 (17H, m, Boc, Lys b,c,d-H6, Gln b-
H2), 1.76 (1H, m, Leu c-H), 1.95 (1H, m, Leu b-H),
2.08–2.20 (2H, m, Val b-H, Leu b-H), 2.35 (2H, t,
J = 7.4 Hz, Gln c-H2), 3.04 (2H, t, J = 6.6 Hz, Lys
e-H2), 3.37 (1H, t, J = 6.6 Hz, Lys a-H), 3.72 (3H, s,
OMe), 4.31 (1H, d, J = 5.9 Hz, Val a-H), 4.38 (1H, t,
J = 7.4, Gln a-H), 4.45 (1H, dd, J = 8.9, 5.1 Hz, Leu
a-H); MS m/z 601.3948 (M+H) (C28H53N6O8 requires
601.3925).
37 ꢁC. The mixtures were incubated for the appropriate
time periods before the enzymic reaction was stopped by
precipitation of the protein by addition of ZnCl2 to a fi-
nal concentration of 10 mM. The composition of the
mixture was then analysed by HPLC, using the same
mobile phase and conditions as in our previous paper.1
The retention times of the product peaks were compared
with those of synthetic samples.
Acknowledgments
We thank Dr. Steven J. Black and Dr. Timothy Wood-
man (University of Bath) for the NMR spectra and the
EPSRC Mass Spectrometry Centre (Swansea) for some
of the mass spectra. We are very grateful to the Associ-
ation for International Cancer Research for generous
funding in support of the project.
5.38. N-(N-(N-(Ne-(1,1-Dimethylethoxycarbonyl)-Na-
(O-(1,1-dimethylethyl)-N-(phenylmethoxycarbonyl)seri-
nyl)lysyl)leucyl)glutaminyl)valine methyl ester (57)
CbzSer(But)OSu (19 mg, 48 lmol) in dry THF (700 lL)
was stirred with 56 (29 mg, 48 lmol), Et3N (9.8 mg,
96 lmol) and DMAP (1 mg) in dry DMF (300 lL) at
0 ꢁC for 30 min and at 20 ꢁC for 72 h. The evaporation
residue, in EtOAc, was washed with cold aq citric acid
(5%) and H2O and was dried to afford 57 (30 mg, 68%)
as a white solid: mp 234–236 ꢁC; NMR ((CD3)2SO) dH
0.83–0.87 (12H, m, 2· Val-Me, 2· Leu-Me), 1.10 (9H, s,
CH2OBut), 1.25–2.11 (21H, Boc, Val b-H, Gln b-H2,
Leu b,c-H3, Lys b,c,d-H6), 2.85 (2H, t, J = 6.2 Hz, Gln
c-H2), 3.32 (2H, m, CH2OBut), 3.44 (2H, t, J = 5.5 Hz,
Lys e-H2), 3.63 (3H, s, OMe), 4.10–4.24 (5H, Val a-H,
Gln a-H, Leu a-H, Lys a-H, Ser a-H), 5.02 (1H, d,
J = 12.5 Hz) and 5.06 (1H, d, J = 12.5 Hz) (CH2Ph),
6.72–8.06 (13H, m, 8· NH, Ph-H5); MS m/z 878.5264
(M+H) (C43H72N7O12 requires 878.5239).
References and notes
1. Suaifan, G. A. R. Y.; Mahon, M. F.; Arafat, T.;
Threadgill, M. D. Tetrahedron 2006, 62, 11245.
2. Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.; Dos
Santos, S.; Mandal, B.; Mimna, R.; Murat, K.; Patiny, L.;
`
Saucede, L.; Tuchscherer, G. Angew. Chem. Int. Ed. Engl.
2004, 43, 4172.
´
3. Alonso De Diego, S. A.; Mun˜oz, P.; Gonzalez-Mun˜iz, R.;
Herranz, R.; Martın-Martınez, M.; Cenarruzabeitia, E.;
´
´
´
´
´
Frechilla, D.; Del Rıo, J.; Jimeno, M. L.; Garcıa-Lopez,
M. T. Bioorg. Med. Chem. Lett. 2005, 2279.
4. Wittelsberger, A.; Patiny, L.; Slaninova, J.; Barberis, C.;
Mutter, M. J. Med. Chem. 2005, 48, 6553.
5. Chierici, S.; Jourdan, M.; Figuet, M.; Dumy, P. Org.
Biomol. Chem. 2004, 2, 2437.
6. Keller, M.; Boissard, C.; Patiny, L.; Chung, N. A.;
Lemieux, C.; Mutter, M.; Schiller, P. W. J. Med. Chem.
2001, 44, 3896.
5.39. N-(N-(N-(Na-(N-(Phenylmethoxycarbonyl)seri-
nyl)lysyl)leucyl)glutaminyl)valine methyl ester trifluoro-
acetate salt (58)
7. Beck, B.; Hess, S.; Do¨mling, A. Bioorg. Med. Chem. Lett.
2000, 10, 1701.
8. Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.;
Fischer, G. S.; Mutter, M. J. Am. Chem. Soc. 1998, 120,
2714.
9. Campiglia, P.; Gomez-Monterrey, I.; Carotenuto, A.;
Lama, T.; Diurno, M. V.; Bertamino, A.; Mazzoni, O.;
Sala, M.; Novellino, E.; Grieco, P. Lett. Org. Chem. 2006,
3, 539.
10. Abedini, A.; Raleigh, D. P. Org. Lett. 2005, 7, 693.
11. Wo¨hr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.;
Sun, X. C.; Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218.
12. Sampson, W. R.; Patsiouras, H.; Ede, N. J. J. Pept. Sci.
1999, 5, 403.
13. Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wo¨hr,
T.; Mutter, M. J. Am. Chem. Soc. 1997, 119, 918.
14. Denmeade, S. R.; Nagy, A.; Gao, J.; Lilja, H.; Schally, A.
V.; Isaacs, J. T. Cancer Res. 1998, 58, 2537.
15. Loadman, P. M.; Bibby, M. C.; Double, J. A.; Al-
Shakhaa, W. M.; Duncan, R. Clin. Cancer Res. 1999, 5,
3682.
16. Denmeade, S. R.; Jakobsen, C. M.; Janssen, S.; Khan, S.
R.; Garrett, E. S.; Lilja, H.; Christensen, S. B.; Isaacs, J. T.
J. Natl. Cancer Inst. 2003, 95, 990.
Compound 57 (3.0 mg, 3.4 lmol) was stirred in
CF3CO2H (300 lL) and CH2Cl2 (700 lL) for 4 h. Evapo-
ration afforded 58 (quant.) as a highly hygroscopic col-
ourless gum: NMR ((CD3)2SO) dH 0.83 (3H, d,
J = 6.6 Hz, Val-Me), 0.87 (3H, d, J = 6.2 Hz, Leu-Me),
0.89 (6H, d, J = 5.9 Hz) (Val-Me, Leu-Me), 1.25–1.80
(10H, m, Lys b,c,d-H6, Leu b-H2, Gln b-H, Val b-H),
1.85 (1H, m, Gln b-H), 2.04 (1H, nonet, J = 6.2 Hz, Leu
c-H), 2.11 (2H, br t, J = 7 Hz, Gln c-H2), 2.85 (2H, m,
Lys e-H2), 3.57 (2H, br d, J = 5.5 Hz, Ser b-H2), 3.63
(3H, s, CO2Me), 4.11 (1H, br q, J = 7.4 Hz, Ser a-H),
4.15 (1H, dd, J = 8.1, 6.2 Hz), 4.20–4.35 (3H, m, Gln a-
H, Leu a-H, Lys a-H), 5.00 (1H, d, J = 12.5 Hz) and
5.04 (1H, d, J = 12.5 Hz) (CH2Ph), 6.81 (1H, s), 7.28
(1H, s) (CONH2), 7.32 (6H, m, Ser NH, Ph-H5), 7.65
(3H, br, NH3), 7.91 (1H, d, J = 5.9 Hz, NH), 7.93 (1H,
d, J = 7.4 Hz, NH), 8.04 (1H, d, J = 7.8 Hz, Val NH),
8.10 (1H, d, J = 8.2 Hz, NH); MS m/z 722.4099 (M+H)
(C34H56N7O10 requires 722.4089).
5.40. Incubations of peptide constructs with PSA
17. DeFeo-Jones, D.; Garsky, V. M.; Wong, B. K.; Feng, D.-
M.; Bolyar, T.; Haskell, K.; Kiefer, D. M.; Leander, K.;
McAvoy, E.; Lumma, P.; Wai, J.; Senderak, E. T.; Motzel,
S. L.; Keenan, K.; Van Zwieten, M.; Lin, J. H.; Frieding-
The peptide constructs 47, 51 and 58 were incubated
with PSA at a molar ratio of 100:1 in a buffer containing
Tris–HCl (50 mM) and NaCl (140 mM) at pH 7.4 at

3488
G. A. R. Y. Suaifan et al. / Bioorg. Med. Chem. 15 (2007) 3474–3488
er, R.; Huff, J.; Oliff, A.; Jones, R. E. Nat. Med. 2000, 6,
1248.
18. Wo¨hr, T.; Mutter, M. Tetrahedron Lett. 1995, 36, 3847.
19. Guichou, J.-F.; Patiny, L.; Mutter, M. Tetrahedron Lett.
2002, 43, 4389.
25. Green, M.; Berman, J. Tetrahedron Lett. 1990, 31, 5851.
26. Fry, E. M. J. Org. Chem. 1950, 15, 438.
27. Curtius, T. J. Prakt. Chem. 1917, 95, 340.
28. Narita, M.; Ogura, T.; Sato, K.; Honda, S. Bull. Chem.
Soc. Jpn. 1986, 59, 2433.
20. Menger, F. M.; Caran, K. L. J. Am. Chem. Soc. 2000, 122,
11679.
29. Mizutani, M.; Sanemitsu, Y.; Tamaru, Y.; Yoshida, Z. J.
Org. Chem. 1985, 50, 764.
21. Carpino, L. A.; Lonescu, D.; El-Faham, A.; Beyermann,
M.; Henklein, P.; Hanay, C.; Wenschuh, H.; Bienert, M.
Org. Lett. 2003, 5, 975.
22. Ohta, T.; Hosoi, A.; Kimura, T.; Nozoe, S. Chem. Lett.
1987, 10, 2091.
´
23. Ezquerra, J.; de Mendoza, J.; Pedregal, C.; Ramırez, C.
Tetrahedron Lett. 1992, 33, 5589.
24. Molina, M. T.; Valle, C.; Escribano, A. M.; Ezquerra, J.;
Pedregal, C. Tetrahedron 1993, 49, 3801.
30. Griffing, J. M.; Salisbury, L. F. J. Am. Chem. Soc. 1948,
70, 3416.
´
31. Betsbrugge, J. V.; Nest, W. V. D.; Verheyden, P.; Tourwe,
D. Tetrahedron 1998, 54, 1753.
32. Bernardi, F.; Garavelli, M.; Scatizzi, M.; Tomasini, C.;
Trigari, V.; Crisma, M.; Formaggio, F.; Peggion, C.;
Toniolo, C. Chem. Eur. J. 2002, 8, 2516.
33. Green, M.; Berman, J. Tetrahedron Lett. 1990, 31,
5851.