Effects of steric bulk and stereochemistry on the rates of?diketopiperazine formation from N-aminoacyl-2,2-dimethylthiazolidine-4-carboxamides (Dmt dipeptide amides)-a model for a new prodrug linker system

Article abstract of DOI:10.1016/j.tet.2006.09.009

A peptide-like self-immolative molecular clip is required for release of active drugs from prodrugs by endopeptidases. Upon cleavage from the carrier, this clip must collapse and release the drug rapidly. A series of aminoacyl-5,5-dimethylthiaproline (Aaa

Full text of DOI:10.1016/j.tet.2006.09.009

Tetrahedron 62 (2006) 11245–11266
Effects of steric bulk and stereochemistry on the rates
of diketopiperazine formation from N-aminoacyl-
2,2-dimethylthiazolidine-4-carboxamides (Dmt dipeptide
amides)—a model for a new prodrug linker system
Ghadeer A. R. Y. Suaifan,a,y Mary F. Mahon,b Tawfiq Arafatc and Michael D. Threadgilla,
*
aDepartment of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
bX-ray Crystallographic Unit, Department of Chemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
cFaculty of Pharmacy and Medical Technology, University of Petra, Queen Alia Airport Road, 11196 Amman, Jordan
Received 28 June 2006; revised 9 August 2006; accepted 1 September 2006
Available online 10 October 2006
Abstract—A peptide-like self-immolative molecular clip is required for release of active drugs from prodrugs by endopeptidases. Upon
cleavage from the carrier, this clip must collapse and release the drug rapidly. A series of aminoacyl-5,5-dimethylthiaproline (Aaa-Dmt)
N-(2-(4-nitrophenyl)ethyl)amides were designed. Boc-L-aminoacyl fluorides were coupled with R-DmtOH to give Boc-L-Aaa-R-DmtOH,
which were converted to the Boc-L-Aaa-R-Dmt N-(2-(4-nitrophenyl)ethyl)amides. The L,S diastereomeric series was prepared by the reaction
of Boc-Aaa PFP esters with S-DmtOH. The L-Aaa-Dmt N-(2-(4-nitrophenyl)ethyl)amides were allowed to cyclise to diketopiperazines
(DKPs) in aqueous buffers, expelling 2-(4-nitrophenyl)ethylamine as a model for amine-containing drugs. Reaction rates were dependant on
pH. In the L,R diastereomeric series, increasing steric bulk of the Aaa side-chain (Gly, Ala, Phe, Val) led to decrease in the reaction rate. How-
ever, in the L,S series, the greatest rate of reaction was observed for the most bulky amino-acid (Val), with t ¼15 min at pH 8.0. The effects of
½
steric bulk and stereochemistry are rationalised through conformational analysis (NMR and X-ray crystallography) of the starting dipeptide
amides, the product diketopiperazines and key analogues. Since the dipeptides are (almost) exclusively in the cis-amide conformation, trans–
cis interconversion is not relevant. The data suggest that steric interactions in the reacting conformations of the dipeptide amides, as they form
the tetrahedral intermediates, are the controlling factors. Thus, L-Aaa-S-Dmt amides are shown to be excellent candidates for incorporation
into the design of novel prodrugs.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
bipartite or tripartite in concept (Scheme 1). In bipartite pro-
drugs, the trigger (which is the masking group)2 is joined
directly to the effector (the drug moiety to be released);
the activating enzyme modifies the trigger chemically, caus-
ing the effector to be released. Of course, the design of
bipartite prodrugs relies on the possibility of appropriate
modes of attachment of the trigger to the effector. Several
reductively activated prodrug systems work in this way.3,4
Where this is not possible, a linker is interposed between
the trigger unit and the effector, making a tripartite prodrug
(Scheme 1). In this system, the activating enzyme triggers
release of the linker–effector unit; the linker then de-
composes spontaneously to expel the effector (drug). Such
linkers can be as simple as a carbamate ester,5 providing
a carbamate anion as a good leaving group from the trigger;
carbon dioxide is then lost rapidly to give the amine drug.
4-Aminobenzyl carbamate esters are also useful linkers.6
Prodrugs have an increasingly important role in the selective
delivery of potent drugs to their intended site of action in the
body. In a prodrug, a part or the whole of the pharmacophore
is masked by a group, which is removed, usually by the
catalytic activity of an enzyme, in the target site. The utility
of the prodrug approach can be to increase the concentration
of the active drug in the target tissue or to diminish the con-
centration in an organ to which the drug is toxic. The mask-
ing group can also carry functionality to increase aqueous
solubility or to modify the biodistribution of the prodrug.
For example, macromolecular prodrugs, in which many
molecules of the drug are attached to a soluble polymer,
are selectively retained in solid tumours owing to leaky vas-
culature and poor lymphatic drainage.1 Prodrugs can be
* Corresponding author. Tel.: +44 1225 386840; fax: +44 1225 386114;
e-mail: m.d.threadgill@bath.ac.uk
Present address: Faculty of Pharmacy, University of Jordan, Amman,
Peptidasespresent particular problemsas activating enzymes,
in terms of prodrug design in general and linker design in
particular. Exopeptidases, particularly carboxypeptidases,
y
Jordan.
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.09.009

11246
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
Bipartite prodrug system
Enzymatic
activation
Trigger
Effector
Trigger
Effector
Tripartite prodrug system
Enzymatic
activation
Trigger
Linker
Effector
Trigger
Linker
Effector
Effector
Self-immolation of linker
Linker co-
product
Scheme 1. Schematic representation of the release of drugs from bipartite and tripartite prodrugs systems. The trigger or the trigger–linker unit, respectively,
comprises the group which masks the pharmacophore of the drug (effector).
can release amine-containing drugs directly from amides at
the C-terminal of short peptides; for example, doxorubicin
is released from the polymeric prodrug PK1 (in which it is
linked to the polymeric backbone through the sequence
GlyPheLeuGly) by cathepsin B.7 However, some activating
enzymes are endopeptidases, cleaving only amide bonds
between amino acids. In several cases, this leads to the re-
lease of a drug molecule still carrying one or more amino-
acyl unit; this may or may not be deleterious to the desired
pharmacological activity. For example, prodrug 1 is cleaved
by prostate-specific antigen (PSA) in malignant prostate tis-
sue to release a thapsigargin analogue still carrying a Leu
residue,8 as shown in Scheme 2. Similarly, PSA-mediated
cleavage of prodrug 2 releases leucyl-doxorubicin in prostate
tumours.9,10 Interestingly, PSA does not release doxorubicin
from HisSerSerLysLeuGln-doxorubicin. In both cases, the
released Leu-drug construct does have cytotoxic activity.
To address this problem for a drug in which a pendant Leu
would be deleterious to activity, prodrug 3 (Scheme 2) has
been developed.11 PSA causes cleavage of the Ser–Ser
bond, releasing SerPro-vinblastine 4. The N-terminal pri-
mary amine of this dipeptide ester then attacks the ester
carbonyl, forming a diketopiperazine 5 and expelling the
cytotoxic drug 6. Similarly, dipeptide esters 7 of paracetamol
have been proposed as prodrugs to release the analgesic 9
slowly by forming diketopiperazines 8.12
In both of these systems where a dipeptide is the linker, the
drug is a good leaving group, forming either an alkyl ester or
a phenyl ester. Only a limited number of drugs carry an alco-
hol in the pharmacophore, whereas many carry a primary
or secondary amine. Design of a linker where the drug is
AcO
O
O2CC7H15
O
1
N
O
O
O
HO
H
NH
H
O
HO
MuHisSerSerLysLeuGln
N
OH
Et
O
HN
MeO
HO
HO
O
OH
O
O
H
CO2Me
2
N
MeN
O
OH
OMe
O
O
MeO2C
HisSerSerLysLeuGln
N
H
N
HO
Et
H
O
O
AcHypSerSerChgGlnSerSer
HO
NH
N
NH2
NHAc
R’
O
3
OH
O
O
O
N
H
R
6 Vinblastine-OH
+
7
Vinblastine
O
O
H
O
NH2
NHAc
O
N
R
O
H
H
N
+
N
HO
R’
N
H
HO
N
OH
O
O
8
9
5
4
Point of cleavage by PSA
Scheme 2. Examples of prodrugs triggered by an endopeptidase. The thapsigargin analogue prodrug 1 and the leucyl-doxorubicin prodrug 2 both release drug
carrying one aminoacyl unit upon cleavage by prostate-specific antigen (PSA). PSA-mediated cleavage of the vinblastine prodrug 3 releases the Ser-Pro vin-
blastine ester 4, which cyclises to form the diketopiperazine 5, expelling the vinblastine alcohol 6. The prodrug system 7 cyclises spontaneously, forming the
diketopiperazines 8 and expelling paracetamol 9.

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11247
attached to the C-terminus of a dipeptide through an amide
is more challenging, owing to the much lower electrophilic
reactivity of amides. In this paper, we report our develop-
ment of a dipeptide linker suitable for amine-containing
drugs.
5,5-dimethylproline and by Dmt has been exploited in study-
ing the biologically active conformations of peptides.19–26
Since peptides containing Dmt exist mainly in the cis (Z) ter-
tiary amide rotamer, it is likely that aminoacyl-Dmt amides
also adopt predominantly this conformation. We, therefore,
postulated that, despite the lower electrophilicity of amides,
these aminoacyl-Dmt amides should cyclise, expelling the
amine (drug). To test this hypothesis and to study the effects
of relative stereochemistry and steric bulk of the amino-acid
side-chain on the rate of cyclisation and release, a series of
N-terminal-protected aminoacyl-Dmt dipeptide amides
were designed. In these dipeptide amides, 2-(4-nitrophenyl)-
ethylamine was used as a simple model for drugs containing
primary aliphatic amines. The protecting group was chosen
to be Boc, which could be removed under acidic conditions,
which prevent cyclisation. The dipeptide amide salts could
then be placed in aqueous buffers of various pH values and
the rates of cyclisation and release of the model drug from
these candidate prodrug linkers could be measured.
2. Design of the dipeptide amide linker
In peptides with free N-terminal amines, the N-terminal
amino-acid pair can cyclise slowly to give a diketopiperazine
(DKP); the C-terminal remainder of the peptide (an amine
leaving group) is expelled. The rate of DKP formation
depends on the proportion of the dipeptide in the reacting
cis conformation.13 However, most peptide sequences adopt
only the trans conformation, because it is energetically
favourable. The presence of proline at the penultimate posi-
tion greatly enhances the rate of DKP formation and expul-
sion of the amine leaving group, owing to the greater
propensity to adopt a cis-amide conformation (AlaPro is
13% cis13,14). This phenomenon was exploited in the SerPro
linker in prodrug 3; replacement of Pro with acyclic amino
acids suppressed release of vinblastine.11
3. Chemical synthesis
Interestingly, replacement of Pro with 2,2-dimethylthiazoli-
dine-4-carboxylic acid (Dmt) has been reported to force
peptides into 100% cis conformation,14,15 putatively owing
to the major steric clash between the side-chain of the
N-terminal amino-acid and the geminal dimethyl unit. Since
the conformation, and thus biological activity of peptides,
is critically dependant on whether amino-acyl prolines are
in the cis or trans amide rotamer, there exist several
enzymes, which catalyse the interconversion, including
cyclophilins and Pin1.16–18 Control of conformation by
The synthetic approach to the first diastereomeric series of
target dipeptide amide salts 21a–d is shown in Scheme 3.
In this series, L-amino acids carrying side chains with diverse
steric bulk (Gly, L-Ala, L-Val, L-Phe) were coupled to R-2,2-
dimethylthiazolidine-4-carboxylic acid derivatives. Firstly,
2,2-dimethylthiazolidine-4-carboxylic acid 11 (R-Dmt) was
prepared in good yield from condensation of L-Cys hydro-
chloride 10 with 2,2-dimethoxypropane by the method of
Kemp and Carey.27 Initially, it was planned to carry out the
peptide synthesis in the conventional manner for dipeptide
HS
S
S
Me
Me
Cl
Me
Me
i
Cl
ii
N
N
CO2H
CO2R
H3N
CO2H
H2
11
Boc
10
12: R = H
iii
13: R = PFP
v
iv
O
S
N
S
N
S
iv
Me
Me
Me
Me
Me
Me
H
N
H
N
N
CO2R
NHBoc
Boc
O
O
O
NHBoc
NO2
NO2
R'
R'
R'
18a-d: R = H
20a-d
14
vi
iii
19a-d: R = PFP
vi
vii
S
S
N
S
Me
Me
Me
Me
Me
Me
H
H
N
N
N
N
CO2PFP
H2
O
O
O
NH3
O
NH3
NO2
NO2
R'
22b-d
Cl
O2CCF3
21a-d
O2CCF3
15
vii
R'
R'
Me
Me
O
S
N
ix
F
BocHN
CO2H
BocHN
17a-d
H
O
O
N
23b-d
1
6a-d
R'
H
Scheme 3. Synthesis of dipeptide N-(2-(4-nitrophenyl)ethyl)amides 21a–d and diketopiperazines 23b–d. PFP¼pentafluorophenyl. Reagents and conditions:
(i) (MeO)2CMe2, acetone, N2, D; (ii) Boc2O, Pri2NEt, MeCN; (iii) PFPOH, DCC, EtOAc, N2; (iv) 2-(4-nitrophenyl)ethylamine hydrochloride (24), Et3N,
CH2Cl2; (v) 17a–d, Pri2NEt, DMF; (vi) CF3CO2H, CH2Cl2; (vii) HCl, CH2Cl2; (viii) Et3N, CH2Cl2; (ix) 2,4,6-trifluoro-1,3,5-triazine, pyridine, CH2Cl2.

11248
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
amides, by forming the C-terminal amide at the beginning
of the synthesis. Protection of the secondary amine of 11
with Boc proceeded in poor yield, owing to the presence
of three bulky substituents on the carbons adjacent to
this nitrogen. Boc-R-DmtOH 12 was then activated as
its pentafluorophenyl ester 13 for coupling with 2-(4-
nitrophenyl)ethylamine 24 to form the required BocDmt
N-(2-(4-nitrophenyl)ethyl)amide 14 in excellent yield.
Removal of the Boc protection afforded the Dmt N-(2-(4-
nitrophenyl)ethyl)amide salt 15 but all attempts to couple
this with activated amino-acid derivatives led to opening
of the thiazolidine ring, shown by observation of cysteinyl
peptides in the crude multi-component reaction mixtures.
of the molar ratio of diastereoisomers at each step in the syn-
thetic sequence would reveal and quantify any significant
loss of stereochemical integrity. As shown in Scheme 4,
the acid fluorides of a mixture comprising 40% Boc-L-
AlaOH 16b and 60% Boc-D-Ala 25 were prepared as above.
This mixture of enantiomeric acyl fluorides 17b and 26 was
coupled with homochiral R-Dmt 11 to give a mixture of the
L,R product 18b and the D,R product 27 in 36% yield. As ex-
pected, the 1H NMR spectrum of the mixture showed distinct
sets of signals for the two diastereoisomers, in the molar
ratio 2:3, respectively, without prior chromatographic puri-
1
fication. The H–1H COSY spectrum of this mixture high-
lighted these separate sets of signals, confirming that the
diastereoisomers could be distinguished by this technique.
Notably, only one set of signals corresponded to the product
18b derived from homochiral Boc-L-AlaOH 16b alone, con-
firming that the sets of signals in the spectra of the latter did
correspond to rotamers and not to diastereoisomers. Further-
more, the similarity of the ratio of products to the ratio of
starting Boc-Ala enantiomers confirms that there is little or
no diastereoselectivity in the coupling reaction, despite the
modest yield. Moving forward in the sequence, the mixture
of 18b and 27 was converted to a mixture of the correspond-
ing pentafluorophenyl esters 19b and 28 in high yield. The
In view of the difficulty of achieving couplings to Dmt with
the C-terminal amide already in place, an alternative strategy
was investigated, coupling of N-protected amino acids with
11, followed by installation of the N-(2-(4-nitrophenyl)ethyl)-
amide. As noted above, the secondary amine of 11 is severely
sterically hindered and is also deactivated as a nucleophile by
the carboxylate and by the sulfur. Thus, a reactive and steri-
€
cally small acylating agent is required for coupling. Wohr
et al.28 have successfully coupled Fmoc-protected amino-
acyl fluorides with 11 in moderate-to-good yields. Adapting
this procedure to the required Boc-protected analogues,
BocGlyOH 16a, Boc-L-AlaOH 16b, Boc-L-ValOH 16c and
Boc-L-PheOH 16d were converted to the corresponding
acyl fluorides 17a–d, respectively (Scheme 3) by treatment
with cyanuric fluoride in the presence of pyridine. These
acyl fluorides proved to be unstable and were used immedi-
ately in crude form for reaction with 11 in dry DMF. The
Boc-protected dipeptides 18a–d were obtained in 27–35%
yields after careful chromatography. Activation as the
pentafluorophenyl esters 19a–d and coupling with 2-(4-
nitrophenyl)ethylamine then efficiently afforded the required
Boc-protected amino-acyl Dmt N-(2-(4-nitrophenyl)ethyl)-
amides 20a–d. Brief treatment with trifluoroacetic acid in
dichloromethane then afforded the amino-acyl Dmt N-(2-
(4-nitrophenyl)ethyl)amide salts 21a–d, which were suffi-
ciently stable to be stored prior to the cyclisation rate studies.
Me
CO2H
16b
Me
CO2H
25
+
BocHN
BocHN
i
Me
Me
F
F
BocHN
S
BocHN
S
+
+
O
O
26
17b
ii
Me
Me
Me
Me
N
N
CO2H
CO2H
O
O
NHBoc
NHBoc
Me
Me
18b
27
iii
S
N
S
N
Me
Me
Me
Me
+
CO2PFP
CO2PFP
NHBoc
The diketopiperazines 23b–d were also required both as
HPLC analytical standards for the kinetics studies of cycli-
sation and as compounds in their own right for study of their
conformations, to aid in understanding the relative cyclisa-
tion reaction rates. Treatment of the Boc-dipeptide penta-
fluorophenyl esters 19b–d with hydrogen chloride removed
the Boc protection, giving the dipeptide PFP ester salts
22b–d. Since all these carry the excellent pentafluorophen-
oxy leaving group, the corresponding free bases cyclised
very rapidly to afford the required diketopiperazines 23b–d
in good yields.
O
O
NHBoc
Me
Me
19b
28
iv
S
N
Me
Me
H
N
20b
O
O
NHBoc
NO2
NO2
Me
+
S
N
Me
Me
H
N
29
O
O
NHBoc
To confirm that the two sets of signals seen in many of the
NMR spectra did indeed correspond to rotamers about the
tertiary amide bond and did not indicate racemisation of
the N-terminal acyl fluoride prior to coupling, one synthetic
sequence was conducted with a mixture of enantiomers of
BocAla of known composition. This series of experiments
would lead to mixtures of diastereoisomers; firstly, the
NMR spectra of the individual diastereoisomers could be
observed and compared with the spectra from the series start-
ing with homochiral Boc-L-Ala and secondly, examination
Me
Scheme 4. Experiments to demonstrate that no racemisation and no dia-
stereomeric induction took place during the acyl fluoride coupling and sub-
sequent steps towards the dipeptide N-(2-(4-nitrophenyl)ethyl)amides.
PFP¼pentafluorophenyl. The molar ratio of enantiomeric starting materials
BocAlaOH 16b/25 was 2:3 in the mixture; molar ratios of diastereomeric
pairs of intermediates and products 18b/27, 19b/28 and 20b/29 were 2:3
in the mixtures, as determined by 1H NMR. Reagents and conditions:
(i) 2,4,6-trifluoro-1,3,5-triazine, pyridine, CH2Cl2; (ii) 11, Pri2NEt, DMF;
(iii) PFPOH, DCC, EtOAc, N2; (iv) 2-(4-nitrophenyl)ethylamine hydro-
chloride, Et3N, CH2Cl2.

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11249
19F NMR spectrum of this mixture was complex and indi-
cated the presence of two conformers for each of the two
diastereoisomers, with a diastereomeric ratio of 2:3 again.
In the light of the relatively higher yields obtained for cou-
plings of BocVal to the Dmt unit in this diastereomeric
series, couplingswith more sterically demanding pentafluoro-
phenyl esters were explored. As a model, Fmoc-L-ValOH 42
was converted to its pentafluorophenyl ester 43, which was
coupled effectively with 36 to give Fmoc-L-Val-S-DmtOH
44. Similarly, Boc-L-LeuOPFP 45 reacted smoothly with
36 to afford 37e, from which the dipeptide pentafluorophenyl
ester 38e was readily obtained. As with the lower homologue
38c, this material could be converted to the corresponding
DKP 41e and, through the Boc-dipeptide amide 39e, to the
target L-Leu-S-Dmt amide salt 40e.
1
A similar ratio was seen in the H NMR spectrum. These
active esters then reacted with 2-(4-nitrophenyl)ethylamine
24 in the usual way to give a 2:3 mixture of 20b and 29.
This series of experiments did show that the diastereo-
isomers were distinguishable by NMR at each stage and
that little or no racemisation had taken place.
Scheme 5 shows the synthetic approaches to the two target
dipeptide amides 40c and 40e in the L,S diastereomeric
series. As a model experiment, D-ValOH 16c was converted
to its acyl fluoride 17c, which was coupled with 11 in the
usual way to give Boc-D-Val-R-DmtOH 32 in 50% yield.
This dipeptide was then taken through to the pentafluoro-
phenyl ester 33 and the Boc-protected dipeptide amide 34
in the usual way. In the target L,S series, condensation of
D-Cys 35 with 2,2-dimethoxypropane gave S-DmtOH 36
and coupling with Boc-L-Val acyl fluoride 17c gave the
L,S-Boc-dipeptide 37c in 35% yield. The corresponding
pentafluorophenyl ester 38c was then converted to the
DKP 41c, by deprotection and treatment with base, and to
the protected dipeptide amide 39c and through to the target
L-Val-S-Dmt dipeptide amide salt 40c, as for the L,R dia-
stereoisomer 21c, above. As expected, the 1H NMR spectra
of the enantiomers 34 and 39c were identical.
4. DKP formation studies
The rates of cyclisation of the dipeptide amides 21a–d, 40c
and 40e to form the DKPs 23a–d, 41c and 41e, respectively,
and to expel the model drug 24 (Scheme 6) were studied in
aqueous buffer at pH 8.0 and 7.0, to reflect the physiological
pH range. The reaction of the L-Val-S-Dmt dipeptide amide
40c was also studied at pH 6.0. Experiments were conducted
in duplicate or triplicate at 37 ꢀC. HPLC analysis of the re-
action mixtures was performed at appropriate time points
to enable the determination of the rates of the reactions.
UV detection at 275 nm reported on the loss of the starting
dipeptide amides and production of 24 (through the
Me
Me
Me
Me
Me
Me
FmocHN
COR
BocHN
COR
BocHN
COR
42: R = OH
16e: R = OH
30: R = OH
31: R = F
i
ii
i
43: R = OPFP
45: R = OPFP
iii
S
S
N
Me
Me
Me
Me
H
N
iv
N
COR
O
O
O
NHBoc
NHBoc
NO2
Me
32: R = OH
Me
i
Me
Me
v
34
33: R = OPFP
HS
S
N
S
Me
Me
Me
Me
Cl
vi
Cl
N
H3N
CO2H
CO2H
NHFmoc
CO2H
viii
H2
36
O
35
44
vii
Me
Me
x,xi
Me
S
S
N
S
N
Me
Me
Me
Me
Me
O
xii,xi
H
O
N
COR
COR
O
O
NHBoc
NHBoc
38c
N
H
R'
Me
Me
Me
41c,e
37c: R = OH
37e: R = OH
Me
i
i
38c: R = OPFP
38e: R = OPFP
iv
iv
S
N
S
Me
Me
Me
ix
H
N
H
Me
N
N
O
O
O
O
NH3
NHBoc
NO2
R'
NO2
39c,e
40c,e
R'
O2CCF3
Scheme 5. Synthesis of the S-thiazolidine series of dipeptide amides 40c and 40e and diketopiperazines 41c and 41e. PFP¼pentafluorophenyl. Reagents and
conditions: (i) PFPOH, DCC, EtOAc, N2; (ii) 2,4,6-trifluoro-1,3,5-triazine, pyridine, CH2Cl2; (iii) 11, Pri2NEt, DMF; (iv) 2-(4-nitrophenyl)ethylamine hydro-
chloride, Et3N, CH2Cl2; (v) (MeO)2CMe2, acetone, N2, D; (vi) 43, Pri2NEt, THF, DMF; (vii) 17c, Pr2i NEt, DMF; (viii) 45, Pri2NEt, DMF; (ix) CF3CO2H, CH2Cl2;
(x) HCl, CH2Cl2; (xi) Et3N, CH2Cl2; (xii) CF3CO2H.

11250
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
Me
Me
S
S
N
Me
Me
H
N
i
H2N
H
N
+
O
O
O
O
O
N
NO2
NH3
NO2
R'
H
H
R'
R'
O2CCF3
21a-d
23a-d
24
Me
Me
O
S
N
S
N
Me
Me
H
N
i
H2N
H
+
O
O
NH3
N
NO2
NO2
R'
O2CCF3
40c,e
41c,e
24
Scheme 6. Studies on rates of ring-closure of dipeptide amides 21a–d, 40c and 40e to diketopiperazines 23a–d, 41c and 41e, expelling the model drug
2-(4-nitrophenyl)ethylamine 24. Reagent and conditions: (i) aqueous buffer pH 6.0, 7.0 or 8.0.
Table 1. Half lives of cyclisation of dipeptide N-(2-(4-nitrophenyl)ethyl)-
amides 21a–d, 40c and 40e in aqueous buffer at pH 6.0, 7.0 and 8.0, forming
the corresponding diketopiperazines 23a–d, 41c and 41e and expelling
2-(4-nitrophenyl)ethylamine 24
nitrophenyl chromophore), whereas simultaneous detection
at 225 nm allowed quantification of the product DKPs. Ex-
amples of graphs showing consumption of the starting mate-
rials and formation of the products are shown in Figure 1.
Each reaction followed first-order kinetics closely, as
expected, and the calculated half lives are shown in Table 1.
A wide range of half lives was seen at pH 8.0 (15 min to
28 h) and at pH 7.0 (1 h to >45 h).
Compound R
Stereoisomera
t
½
(37 ꢀC, h)
pH 7.0
pH 8.0
pH 6.0
21a
21b
21c
21d
40c
40e
H
Me
CHMe2
CH2Ph
CHMe2
R
10–11b 42
3–4b 8–9b
26–27b >45
28–29b NDc
NDc
NDc
NDc
NDc
L,R
L,R
L,R
L,S
Comparison of the cyclisation half lives at pH 8.0 with the
corresponding values at pH 7.0 indicates that each dipeptide
amide reacts some 2.5–4 times faster at higher pH. Since,
it is only the unprotonated free-base dipeptide that carries
the nucleophilic primary amine that attacks the secondary
amide carbonyl, this consistently observed effect of pH on
the overall reaction rate reflects the amount of this proto-
tropic form that is present in solution. Since the pKa values
of the N-terminal amines are likely to be ca. 8–9, changing
the pH by one pH unit will have a marked effect on the con-
centration of the free-base form in solution and, thus, on the
reaction rates. In the case of 40c, further lowering the pH to
0.25–0.3b 0.75–1.25b 10
CH2CHMe2 L,S
1.0–1.5b 2.5
NDc
a
L/D refers to the configuration of the N-terminal amino-acid; R/S refers to
the configuration of the thiazolidine.
Range of values from >1 experiment.
Not determined.
b
c
6.0 slows the reaction a further 10-fold, reflecting the very
low concentration of the reactive prototropic form in this
acidic milieu.
In the L,R diastereomeric series, the effect of increasing the
steric bulk of the amino-acid side-chain was not straightfor-
ward. It had been predicted that increasing the bulk would
increase the rate of reaction by increasing the proportion
of the reactive Z tertiary amide rotamer in solution. Compar-
ison of the half-life of cyclisation of the Gly-R-Dmt amide
21a with that of the L-Ala-R-Dmt analogue 21b would sug-
gest that this prediction was true. However, the L-Val-R-Dmt
and L-Phe-R-Dmt dipeptide amides 21c and 21d, containing
the much more bulky isopropyl and benzyl side chains, re-
spectively, cyclise around nine times slower at pH 8.0 than
does the L-Ala-R-Dmt analogue 21b. Thus, with the excep-
tion of the Gly analogue, increase of steric bulk of the
side-chain decreases the reaction rate in this series.
5
A
4
3
2
1
0
0
10
20
30
40
Time (h)
50
60
70
80
16
12
8
B
The converse is seen in other diastereomeric series. In gen-
eral, the L,S dipeptide amides cyclise much faster than do
the L,R analogues. Within the series, the L-Val-S-Dmt dipep-
tide amide 40c reacts some 3–4 times faster than does the
L-Leu-S-Dmt dipeptide amide 40e. The isobutyl group of
the latter, although formally larger than the isopropyl group
of the former, is less sterically demanding at the critical
b-carbon atom. The Gly-R-Dmt dipeptide amide 21a is
also formally a member of this diastereomeric series and
its reaction rates are ca. 40 times slower than those of 40c
and 10–15 times slower than those of 40e. Thus, in this
4
0
0
15
30
45
60
75
90 105 120
Time (min)
21c
24
23c
40c
41c
Figure 1. Examples of graphs obtained for typical experiments measuring
consumption of dipeptide amides 21c (A) and 40c (B), formation of
DKPs 23c and 41a and expulsion of 2-(4-nitrophenyl)ethylamine 24 in
aqueous solution at pH 8.0.

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11251
series, increasing the steric bulk of the side-chain increases
the reaction rate.
correspond to amide rotamers, spectra were run at tempera-
tures ranging from 20 to 100 ꢀC. The energy barrier to rota-
tion proved to be low for this type of restricted amide bond
rotation, with significant broadening of the peaks at 30 ꢀC
and full coalescence to a sharp spectrum at 40 ꢀC.
Clearly, simple consideration of the Z/E tertiary amide
rotamer equilibrium cannot provide the complete rationali-
sation of these effects. A detailed study of conformations of
the dipeptide amides and related precursors was therefore
undertaken, along with examination of conformations of
likely intermediates in the courses of the cyclisations.
Although this study did indicate that there was a strong pref-
erence for one of the rotamers, it did not show whether the
more abundant rotamer was Z or E and identification of
the conformation of the major rotamer required a NOESY
spectrum at 20 ꢀC. Firstly, the peaks of the major rotamer
were fully assigned using a COSY spectrum and some con-
formational information was derived from the coupling con-
stants. Interestingly, the signals for the Gly methylene
protons were widely separated by some 0.47 ppm, which
suggested a conformation in which they were located rela-
tively close in space to the chiral 4-C of the thiazolidine.
In the 2-(4-nitrophenyl)ethyl unit, the protons of the methyl-
ene adjacent to the amide nitrogen were magnetically in-
equivalent (d 3.39 and 3.47), as expected, but the protons
of the more remote methylene resonated as a simple triplet,
reflecting its greater distance from the chiral centre. In the
thiazolidine, a COSY cross-peak was seen between 4-H
and 5b-H (d 3.31) but not between 4-H and 5a-H (d 3.10);
this lack of a cross-peak is consistent with the multiplicity
of the 5a-H signal as a simple doublet, coupling only to its
geminal partner. The lack of observed coupling between
4-H and 5a-H points to a dihedral angle close to 90ꢀ between
these protons. In the NOESY spectrum, a cross-peak was
seen between 4-H and 5a-H but no peak was seen between
4-H and 5b-H, confirming the above assignment. A strong
cross-peak diagnostic for the identity of the major rotamer
was present between 4-H and the downfield Gly methylene
proton, whereas a weak peak was seen between 4-H and
the other Gly methylene proton. Cross peaks between the
geminal dimethyl unit and the Gly methylene protons were
absent, confirming that the glycine was close in space to
the 4-C of the thiazolidine.
5. Conformational studies—dipeptides
5.1. NMR studies—L,R diastereomeric series
1
As expected, the H NMR spectrum of 12 shows the pres-
ence of two rotamers about the Boc–N carbamate bond, in
approximately equal amounts. Figure 2 shows the signals
corresponding to the 4-H of the thiazolidine in (CD3)2SO
at 20 and 80 ꢀC. At lower temperature, the signals for 4-H
are distinct and, at higher temperature, the signals are fully
coalesced.
The dipeptides were designed such that the geminal di-
methyl unit should provide sufficient steric bulk to perturb
the slow equilibrium between the rotamers about the tertiary
amide peptide bond in favour of the cis (Z) conformer, which
is the one required for the formation of the DKPs and ex-
pulsion of the 2-(4-nitrophenyl)ethylamine leaving group.
As an indicator of the populations of the rotamers of the
N-terminal free-base dipeptides (some of which were ex-
pected to cyclise too quickly for satisfactory measurement of
rotamer populations), the ratios of the rotamers of the Boc-
aminoacyl-DMT amides 20a–d, 39c,e were measured by
1H NMR spectroscopy. Firstly, the spectrum (in (CD3)2SO
at 20 ꢀC) of the simplest Boc-dipeptide amide (20a), which
contains glycine and is thus a member of both diastereomeric
series, showed two sharp singlets at d 1.32 and 1.38 in the
approximate ratio of 1:11, corresponding to the protons of
the Boc group. The remainder of the spectrum also showed
evidence for major and minor populations but the peaks were
overlapping, precluding other measurements of the ratio
of rotamers. To confirm that these populations did indeed
Similar conformational studies were carried out on the other
three Boc-protected dipeptide amides in the R-Dmt series.
1
The 1-D H NMR spectra of 20b–d in (CD3)2SO at 20 ꢀC
each showed only one set of signals, indicating the presence
of only one rotamer about the tertiary amide bond in each
case. The identity of the single rotamer of the Ala analogue
20b was established by a NOESYexperiment, which showed
a connectivity between Ala a-H and the thiazolidine 4-H.
Correlations between the geminal methyls and all the pro-
tons of the BocAla moiety were correspondingly absent,
confirming the Z tertiary amide conformation for the sole
rotamer of 20b. The spectra of 20c and 20d were similarly
indicative of the Z rotamers. Thus, all four members of this
L,R diastereomeric series exist either predominantly or
exclusively in this conformation in DMSO solution, which
is the one required for cyclisation.
5.2. Crystal structure of Boc-L-Ala-R-Dmt
N-(2-(4-nitrophenyl)ethyl)amide 20b
Boc-L-Ala-R-DmtNH(CH2)2C6H4NO2 20b formed crystals,
from ethyl acetate/hexane, of a suitable quality for X-ray
crystallography. The crystal structure is shown in Figure 3.
This molecule, which exists in DMSO solution as the Z
20°C
80°C
Figure 2. Parts of 1H NMR spectra of BocDmtOH 12 in (CD3)2SO at 20 and
80 ꢀC.

11252
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
Table 2. 1H NMR chemical shifts of the diastereotopic thiazolidine 5-pro-
tons in the Boc-Aaa-DmtNH(CH2)2C6H4NO2 analogues 20a–d, 34, 39c
and 39e
b
c
Compound Stereoisomera d (5-Htrans
)
d (5-Hcis
)
Dd (5trans-Hꢁ5cis-H)
20a
20b
20c
20d
34
R
3.31
3.24
3.12
2.89
3.36
3.36
3.45
3.10
3.32
3.63
3.20
3.04
3.04
3.05
D0.21
L0.08
L0.41
L0.31
+0.32
L,R
L,R
L,R
D,R
L,S
L,S
39c
39e
+0.32
+0.30
a
L/D refers to the configuration of the N-terminal amino-acid; R/S refers to
the configuration of the thiazolidine.
5-H trans to 4-H.
5-H cis to 4-H.
b
c
latter, owing to the greater distance between the isopropyl
side-chain of the Val and the pseudo-axial 2-methyl group
on the thiazolidine. Also noteworthy is the effect of steric
bulk of the side-chain of the amino-acid on the chemical
shifts of the diastereotopic thiazolidine 5-protons (Table 2).
In the L,R series (20b–d), the simple doublet for 5-H cis to
4-H resonated downfield to the signal for 5-H trans to 4-H
but in the L,S (and D,R) diastereomeric series (34, 39c,
39e), the chemical shifts are reversed; this may reflect a
subtle conformational difference in the thiazolidine ring
between the two series. The chemical shifts of the cor-
responding protons in the Gly analogue 20a resembled those
of the L,S/D,R series.
Figure 3. X-ray crystal structure plot of one moleculewithin the asymmetric
unit of Boc-L-Ala-R-DmtNH(CH2)2C6H4NO2 20b, showing crystallo-
graphic numbering. Ellipsoids are represented at 30% probability and hy-
drogens are omitted for clarity.
rotamer, also adopts this conformation in the solid state. In
addition to any conformational preference, which may be
driven by steric factors, there is an intramolecular hydrogen
bond tying the Boc carbonyl oxygen at the N-terminal of the
dipeptide to the secondary amide N–H at the C-terminal. The
intermolecular hydrogen bonds from the central tertiary
amide carbonyl oxygen to the N-terminal Boc-N–H form a
chain through the crystal.
Examination of the 1H NMR spectra of the dipeptide amide
salts 21a–d showed the presence of only one conformer in
solution; this is presumably the Z tertiary amide rotamer,
in the light of the similarity of the spectra to those of the
N-Boc-protected precursors 20a–d.
5.3. NMR studies—L,R diastereomeric series
NOESY spectroscopy of the dipeptide pentafluorophenyl es-
ter hydrochloride salts 22c and 22d gave some contrasting
conformational information. The 1H NMR spectrum of
22c showed the presence of only one conformer in DMSO
solution. Whereas the corresponding NOESY spectrum did
allow assignment of the signals for the individual 5-H pro-
tons, no cross peaks were seen between signals for protons
on the Val moiety and those on the thiazolidine; thus the sole
rotamer could not be identified. However, in the case of the
Phe analogue 22d, the NOESY spectrum of a DMSO solu-
tion revealed through-space interaction between the upfield
Me (d 1.87) and the ortho-protons on the phenyl group. There
is also connectivity between the downfield Me (d 1.92) and
the Phe a-H (d 4.24). These data suggest that this dipeptide
ester salt may be exclusively in the E amide conformation.
This conformation may be favoured by the considerable
steric bulk of the pentafluorophenyl ester driving the Phe
residue towards the geminal dimethyl unit.
In the L,S diastereomeric series, the conformations of the
tertiary amides of two examples were examined, in addi-
tion to that of BocGly-R-DmtNH(CH2)2C6H4NO2 20a
which, as noted above, can be considered as a member of
both series. 1H NMR spectra of the enantiomers Boc-
D-Val-R-DmtNH(CH2)2C6H4NO2 34 and Boc-L-Val-S-
DmtNH(CH2)2C6H4NO2 39c showed the presence of two
rotamers in the abundance ratio of 4:1; the major rotamer
was again the cis-amide Z conformer, although a variable-
temperature study showed that coalescence of the sets of sig-
nals was not yet complete at the highest temperature studied
(80 ꢀC). The signals for the rotamers of most tertiary amides
coalesce below this temperature and that the current obser-
vation indicates a relatively high energy barrier to rotation
in this molecule. The same ratio of tertiary amide rotamers
was present in the solution of the homologue Boc-L-Leu-D-
DmtNH(CH2)2C6H4NO2 39e.
Thus, the Z/E ratio of rotamers is strongly in favour of
the isomer in each case and the geminal dimethyl unit
at the 4-position of the thiazolidine has been effective
in achieving this desired outcome. However, direct
comparison of the rotamer ratios for the diastereomers
Boc-L-Val-R-DmtNH(CH2)2C6H4NO2 20c and Boc-L-Val-
S-DmtNH(CH2)2C6H4NO2 39c suggests that this sterically
driven bias towards the Z rotamer is not as effective in the
6. Conformational studies—diketopiperazines
As a contribution to understand the marked different rates of
ring-closure to form the DKPs and to expel the 2-(4-nitro-
phenyl)ethylamine, NMR and crystallographic studies were
carried out on the various DKPs. The cyclo-L-Ala-R-Dmt
and cyclo-L-Val-R-Dmt DKPs 23b and 23c were crystalline

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11253
compounds and thus amenable for the determination of
6.2. Crystal structure of cyclo-L-Ala-R-Dmt 23b
conformation in the solid state by X-ray crystallography.
Although there have been several studies reported in the lit-
erature on the conformations of prolyl DKPs,29–32 there are
no reports to date for similar studies with their thiaproline
analogues.
A crystallographic quality crystal of cyclo-L-Ala-R-Dmt 23b
was grown from ethyl acetate/hexane and the X-ray crystal
structure was determined. Figure 4 shows the intermolecular
hydrogen-bonding pattern evident in the crystal and the
structure of a single molecule, together with the crystallo-
graphic numbering scheme. The intermolecular hydrogen-
bonding forms a ribbon through the crystal, with the N–H
being hydrogen-bonded to the corresponding secondary
amide carbonyl of the adjacent molecule. The tertiary amide
carbonyl (from the dimethylthiaproline unit) does not par-
ticipate in hydrogen-bonding. Within an individual molecule
of 23b, the DKP ring is in a flattened boat conformation,
with the thiazolidine in a half-chair. This ring conformation
places the geminal methyl groups in equatorial and axial
positions; the 6-methyl occupies a pseudo-equatorial posi-
tion. Notably, this 6-methyl group is remote in space from
the geminal dimethyl unit and, indeed, from other sterically
bulky groups.
6.1. NMR studies—L,R diastereomeric series
DKP 23d, derived from L-Phe and R-2,2-dimethylthiazoli-
dine-4-carboxylic acid, formed a non-crystallisable gum and
an attempt was made to determine its conformation in solu-
tion by NOESY spectroscopy and by examination of 1H–1H
NMR coupling constants. Although the former was rela-
tively uninformative about conformation, it did serve to
assign the two singlet signals due to the methyl groups and
to distinguish the signals for the two 1-H protons. This
NOESY spectrum showed NOE connectivity between one
1-H double doublet (d 3.26) and the 8a-H (d 4.53); thus
the signal at d 3.26 is due to the 1-H on the lower (a) face
of the molecule. NOE connectivity was also seen between
the upfield methyl singlet at d 1.87 and the b-face 1-H at
d 3.19; thus this upfield singlet signal (d 1.87) is due to the
methyl on the b-face. Another NOE connection was seen
between the ortho-protons of the phenyl group and 6-H, sug-
gesting that the phenyl group was pointing away from the
heterocycle.
NMR observations show that a similar conformation is also
likely to be adopted in solution in chloroform. The coupling
constants between the axial 8a-H and the 1b-H and 1a-H pro-
tons are 3J¼10.2 and 6.6 Hz, respectively. These values are
consistent with the crystallographic dihedral angles (8a-H)-
(8a-C)-(1-C)-(1b-H) of 166ꢀ and (8a-H)-(8a-C)-(1-C)-(1a-H)
of 44ꢀ, according to the Karplus relationship. The axial 6-H
3
The boat conformation of the DKP ring of 23d was con-
firmed by the presence of the five-bond coupling 5J¼
0.8 Hz between the axial 8a-H and the axial 6-H.33 There-
fore, the PhCH2 side-chain is in an equatorial position and
is far from the geminal dimethyl unit. The thiazolidine is
probably in the half-chair conformation, with a trans-diaxial
couples with the adjacent N–H by J¼1.0 Hz, which cor-
responds to the crystallographic dihedral angle of 97ꢀ.
Longer-range couplings are also observed. A four-bond cou-
pling 4J¼1.6 Hz is seen between 8a-H and the NH; the cou-
pling path between these atoms contains four atoms in
one plane and one terminal atom out of that plane, thus the
W-arrangement is almost adopted. A five-bond coupling
3
coupling J¼11.3 Hz between 8a-H and 1b-H. The corre-
3
sponding axial–equatorial coupling to 1a-H is J¼5.9 Hz.
A four-bond coupling 4J¼1.6 Hz is observed between 8a-H
and NH, as seen for 23b. The coupling constants from the
Ph-CH2 protons to 6-H are 3J¼10.2 and 3.9 Hz; thus the Ph
cannot be antiperiplanar to the 6-H, as a coupling constant of
10.2 Hz is only consistent with antiperiplanar H–C–C–H; the
phenyl is either orientated towards the nitrogen or towards
the carbonyl oxygen in a staggered conformation. These
data contrast with those reported33 for the analogous ditryp-
tophenaline, in which the two corresponding coupling con-
3
stants between Ph-CH2 and 6-H are J¼4.4 and 3.1 Hz;
these values are used to support a conformation with Ph lo-
cated over the DKP ring. We have also noted a similar confor-
mation for cyclo-L-Phe-D-Pro.34 However, the data for 23d
are consistent with the X-ray structure-derived conformation
reported35 for cyclo-L-Phe-L-Pro, in which the DKP is boat
conformation, CH2Ph is equatorial and the phenyl is antiperi-
planar to the carbonyl. Further evidence for the boat confor-
mation of the DKP ring is provided by the five-bond coupling
5J¼0.8 Hz between 8a-H and 6-H. Maes et al.33 observed
a similar coupling in ditryptophenaline, a natural product
from Aspergillus flavus var. columnaris, which contains a
cyclo-L-Phe-L-Pro unit fused to a heterocycle through the
Pro side-chain loop, and used it to confirm that the DKP is
in a boat conformation in solution with the corresponding
protons in axial and cis. Thus 8a-H and 6-H in 23d are
also shown to be 1,4-diaxial and cis, an arrangement only
available in a boat conformation.
Figure 4. (Upper) Plot of the asymmetric unit in the X-ray crystal structure
of cyclo-L-Ala-S-Dmt 23b. Ellipsoids are represented at 30% probability.
(Lower) Intermolecular hydrogen-bonding pattern for 23b in the crystal.

11254
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
5J¼0.8 Hz was observedbetween the axial 8a-H and the axial
coupling is seen between 6-H and the adjacent Me2CH; in
the solid state, the corresponding dihedral angle is 70ꢀ. Inter-
estingly, Young et al.32 reported an analogous coupling con-
stant 3J¼2.7 Hz for coupling between the Val a-H and the Val
b-H in cyclo-L-Val-L-Pro, which, together with lanthanide
shift-reagent studies, is interpreted as showing that this
DKP adopts the same conformation with respect to the iso-
propyl side-chain.
6-H, confirming the boat conformation.33
6.3. Crystal structure of cyclo-L-Val-R-Dmt 23c
As for cyclo-L-Ala-R-Dmt 23b, a crystal of cyclo-L-Val-R-
Dmt 23c was grown from ethyl acetate/hexane and the
X-ray crystal structure was determined. Figure 5 shows
the intermolecular hydrogen-bonding pattern evident in the
crystal and the structure of a single molecule, together with
the crystallographic numbering scheme. The intermolecular
hydrogen-bonding motif in this crystal is different from that
in the crystal of cyclo-L-Ala-R-Dmt 23b, above, in that the
hydrogen-bonded pairs are formed between the N–H hydro-
gens of each of a pair of molecules and the secondary amide
carbonyls. Again, the tertiary amide carbonyls do not take
part in hydrogen-bonding.
6.4. NMR studies—L,S diastereomeric series
In the L,S diastereomeric series, neither cyclo-L-Val-S-Dmt
41c nor cyclo-L-Leu-S-Dmt 41e formed crystals of a suitable
quality for crystallography. However, 1H NMR spectroscopy
allowed some inferences to be made about their conforma-
tions in solution.
In contrast to the L,R series, where the coupling constant
between 6-H and the adjacent N–H was very small, this
coupling was much larger in the L,S series, with 3J¼3.5 Hz
for 41c and with 3J¼5.0 Hz for 41e. These values are incon-
sistent with the boat conformations of the DKP rings but
indicate that the DKPs are likely to be flattened from the
boat form. This flattening would relieve steric compres-
sion between the 6-side-chain substituents (Me2HC– or
Me2CHCH2–) and 8a-H. Indeed the coupling constants
show that the flattening is greater in 41c than in 41e, reflect-
ing the greater steric demand of the isopropyl group com-
pared to that of the 2-methylpropyl group. With the DKP
tending towards planarity, the side chains are remote in space
from the geminal dimethyl unit. As expected, 8a-H did not
couple with 6-H in either compound in this series, since
these protons are now trans and no longer 1,4-diaxial. The
conformations of the five-membered rings are likely to be
half chairs, as indicated by the coupling constants between
8a-H (now on the b-face) and 1a-H (10.9 Hz for 41c and
10.5 for 41e) and between 8a-H and 1b-H (3J¼5.5 Hz for
41c and 3J¼5.9 Hz for 41e).
The X-ray crystallographic study of cyclo-L-Val-R-Dmt 23c
shows that this molecule adopts a conformation similar to
that of cyclo-L-Ala-R-Dmt 23b. Again, the DKP ring is in
a boat conformation, with the thiazolidine in a half-chair.
The geminal methyl groups at position-3 are in equatorial
and axial positions; the 6-isopropyl occupies a pseudo-equa-
torial position. This 6-isopropyl group is again remote in
space from the bulky geminal dimethyl unit. The solution
conformation of cyclo-L-Val-R-Dmt 23c in (CD3)2SO is sim-
ilar, as shown by the H NMR spectrum. The J coupling
constants between the axial 8a-H and the 1b-H and 1a-H
are 11.7 and 5.9 Hz, respectively, and are again consistent
with the dihedral angles (8a-H)-(8a-C)-(1-C)-(1b-H) of
169ꢀ and (8a-H)-(8a-C)-(1-C)-(1a-H) of 41ꢀ. In this case,
the coupling between the axial 6-H and the adjacent N–H
is too small to be resolved; the dihedral angle in the crystal
structure is 98ꢀ. The diagnostic five-bond coupling 5J¼
1.2 Hz was again observed between the axial 8a-H and the
axial 6-H, confirming the boat conformation in solution.33
Turning to the conformation of the side-chain, a 3J¼2.3 Hz
1
3
7. Conclusions
At pH 8.0 and 7.0, there are trends relating the bulk of the
side-chain of the N-terminal amino-acid and the rate of
cyclisation. There are also marked differences in the rate
of cyclisation between the diastereoisomers. As shown in
Table 1, in the L,R series, the fastest rates are observed for
L-Ala-R-DmtNH(CH2)2C6H4NO2 21b at both pH values.
Increasing the steric bulk of the side-chain from Me to iso-
propyl or benzyl (in L-Val-R-DmtNH(CH2)2C6H4NO2 21c
or L-Phe-R-DmtNH(CH2)2C6H4NO2 21d, respectively)
slows the cyclisation by ca. seven-fold. Interestingly, the
smallest analogue, Gly-R-DmtNH(CH2)2C6H4NO2 21a,
also cyclised some three-fold slower than did 21b. Scheme 7
shows some of the relevant conformational and prototropic
equilibria involved in the cyclisation, which aid in the ration-
alisation of these observations. Firstly, cyclisation can only
occur from the free-base forms 21A/40A, 21B/40B and
21C/40C, in which the amine is nucleophilic, rather than
from the cationic protonated forms 21D/40D and 21E/
40E. However, the prototropic equilibria B and C would
be rapid and the pKa of 21D/40D and 21E/40E would be ex-
pected to be largely independent of the steric bulk of the
Figure 5. (Upper) Plot of one molecule in the asymmetric unit of cyclo-L-
Val-R-Dmt 23c. Ellipsoids are represented at 30% probability. (Lower)
Intermolecular hydrogen-bonding pattern for 23b in the crystal.

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11255
S
N
S
N
Me
Me
Me
Me
H
N
H
N
A
H3N
O
+
R’
O
O
O
+
O
NH3
NO2
NO2
NO2
R’
R’
21D/40D
21E/40E
C
B
S
N
S
N
S
N
Me
Me
Me
Me
Me
Me
H2N
H
N
H
N
H
N
E
D
O
O
O
R’
O
O
NH2
21A/40A
R’
NO2
NO2
H2N
21B/40B
21C/40C
21cA
40cA
S
N
H
N
Me
Me
H
NO2
OH
O
N
H
R’
47A
46
48A
S
N
Me
Me
H
O
O
N
H
R’
23/41
23cA
41cA
Scheme 7. Cyclisation of dipeptide N-(2-(4-nitrophenyl)ethyl)amides to form diketopiperazines, with expulsion of 24, showing selected conformations and
prototropic equilibria of starting dipeptide amides and reacting conformations of intermediates. The sequence 21A/40A/46/23/41 shows the general course
of the reaction; sequences 21cA/47A/23cA and 40cA/48A/41cA show the specific structures for the diastereoisomers of the L-Val-derived analogues.
Structures 21cA, 47A, 40cA, 48A and 41cA are derived from energy-minimising MM2 calculations; structure 23cA is an X-ray crystal structure. In structures
21cA, 47A, 40cA and 48A, the nitro group has been omitted for clarity.
side-chain R0. Thus, equilibria B and C can be discounted as
sources of the differences in rates of ring-closure. As the
core peptide bond in all the dipeptides is a tertiary amide,
the populations of the Z and E amide rotamers in the reaction
mixtures could be predicted to be an important determinant
of the rate of cyclisation, since only the Z conformer places
the primary amine sufficiently close to the secondary amide
carbonyl carbon, the electrophile in the key step. Since the
rate of interconversion of the tertiary amide rotamers is
relatively slow at ambient temperature, the 2,2-dimethyl-
thiazolidine unit was designed to bias the equilibrium (equi-
librium D, Scheme 7) towards the required Z conformer
21A/40A. As noted above, it was not possible to measure
the populations of the tertiary amide bond rotamers directly
in the free-base dipeptide amides 21A/40A, owing to the
possible perturbation of the equilibrium by the cyclisation
reaction consuming one of the components. However,
1H NMR spectra of the protonated (and thus unreactive)
forms of salts 21a–d showed that each existed solely in the
Z tertiary amide conformation, irrespective of the bulk
of the side-chain R0. Moreover, the state of protonation of
L-Ala-L-ProNH2 has been reported to have little effect on the
cis/trans conformational equilibrium.14 Thus, equilibrium A
is not relevant to the effects of side-chain bulk on the rate of
cyclisation. Similarly, as discussed above, the electrically
neutral N-Boc precursors 20a–d were shown to exist solely
or >90% as the Z tertiary amide rotamers. Taken together,
these conformational data suggest that the free-base di-
peptide amides are likely to be all (almost) exclusively in
the reactive Z tertiary amide conformation.
If the bulk of the side-chain R0 does not influence the rate
of cyclisation by perturbing the Z/E tertiary amide rotamer
population inthestartingmaterials, then thequestionremains
at which point in the cyclisation process does the nature of R0
have its effect? Since the primary amine has to be located
close to the secondary amide carbonyl as the molecules move
towards the first transition state, the conformation about the
carbonyl–Ca bond in the N-terminal amino-acid is also
important, i.e., the dipeptides must adopt conformer 21A
(Scheme 7), rather than conformers such as 21B, in which the
amine is pointing away from the target secondary amide. No
data are available on the populations of these two types of
conformers but one may speculate, on steric grounds, that
the unreactive conformer 21 was more likely when R0¼H
in Gly-R-DmtNH(CH2)2C6H4NO2 21a and the reactive
conformer 21 is more favoured when R0 is large, in L-Ala-
R-DmtNH(CH2)2C6H4NO2 21a, L-Val-R-DmtNH(CH2)2-
C6H4NO2 21c and L-Phe-R-DmtNH(CH2)2C6H4NO2 21d.
This would rationalise theslow cyclisation of theGly analogue.
Now, comparing the rates of formation of DKPs from the
remaining members of the L,R diastereomeric series, is
the steric bulk of R0 exerting its effect on the first step of

11256
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
the reaction, as the primary amine of the reactive conformers
21 attack the secondary amide carbonyl to form the tetra-
hedral intermediates 46 or is this effect evident in the second
step of the reaction, the collapse of the intermediate to form
the DKP 23/41 with expulsion of the leaving group 2-(4-
nitrophenyl)ethylamine 24? To answer this question, the
structures of the key intermediates were compared. Structure
21cA (Scheme 7) is a 3-D representation of the reacting con-
formation of 21c (R0¼isopropyl, derived from L-Val), gener-
ated using a MM2-minimised structure. Similarly, structure
47A is a 3-D representation of the minimised structure of
intermediate 46 (R0¼isopropyl, derived from L-Val) and
structure 23cA is a 3-D representation of the crystal structure
of DKP 23c (R0¼isopropyl, derived from L-Val). Examina-
tion of the DKP structure 23cA, as discussed in detail above,
shows that the isopropyl group is pseudo-equatorial in the
boat conformation of the DKP ring and is thus remote from
the geminal dimethyl unit and, indeed, from all other steri-
cally demanding moieties. The conformations of the other
DKPs in the L,R series are similar. Thus, increasing the steric
bulk of R0 in this series would not cause increase in the steric
crowding in the DKPs 23 and it is therefore highly unlikely
that the effect of bulky R0 on slowing the overall reaction
rate derives influences from the second step, the collapse of
the tetrahedral intermediate. Similarly, the tetrahedral inter-
mediates 46 are shown to have pseudo-chair conformations
for the six-membered ring, as shown in the L-Val-derived
analogue structure 47A. In this conformation, again, the R0
group occupies a sterically open region of space and thus var-
iations in steric bulk of R0 in this intermediate are similarly
unlikely to influence the overall reaction rate significantly.
remote from the methyl groups in intermediate 48A; thus
steric crowding is also unimportant here. However, in the
L,S series, as with the L,R series, it is the steric crowding in
the reacting conformation of the initial dipeptide amide sub-
strate that is critical. As shown in example structure 40cA
(Scheme 7), the reactive conformation of the carbonyl–Ca
bond has the a-hydrogen almost eclipsing the tertiary amide
carbonyl oxygen. This arrangement is clearly much more fa-
vourable on steric grounds than that in the L,R diastereoiso-
mer 21cA, with R0 eclipsing this oxygen. Thus the reacting
conformation is more sterically accessible in the L,S series,
facilitating the rapid formation of the tetrahedral cyclic in-
termediates. Comparing the rates of cyclisation of the
L-Val analogue 40c and the L-Leu analogue 40e, one may
then postulate that the greater steric demand of the tertiary
isopropyl centre may drive the molecule more effectively
into this reacting conformation than does the lesser demand
of the secondary centre of the isobutyl group.
The cyclisation of peptides containing Dmt to give DKPs has
not previously been studied. A related system, the cyclisa-
tion of L-alanyl-L-prolinamide in water to give cyclo-L-
Ala-L-Pro and ammonia, was the subject of a detailed kinetic
analysis by Capasso et al.13 This is a relatively slower
process than the cyclisations of aminoacyl-Dmt amides re-
ported in this paper. In the AlaProNH2 study, it was noted
that the trans/cis conformational interchange of the
tertiary amide was fast36,37 and became rate-limiting only
under conditions were the cyclisation was fast.13 Interest-
ingly, Sager et al.38 note that the cis conformers of pentapep-
tides containing L-Pro or L-Dmt can be readily cyclised to
give cyclic pentapeptides but the corresponding trans con-
formers tend to produce cyclic dimers (cyclic decapeptides)
under the same conditions. Limitation of rate in this confor-
mational interchange step may be important, in principle, in
the cyclisations here, particularly those in the L-Aaa-S-
DmtNH(CH2)2C6H4NO2 diastereomeric series, but it is un-
likely to affect the global rate of reaction since the vast
majority of these reactant molecules are already in the cis
conformation. As expected, pH had a marked effect on the
rate of cyclisation of L-Ala-L-ProNH2, where the trend was
for the reaction to be faster at higher pH.13 However, replot-
ting the data to account for the fraction of the reactive free-
base form in solution indicated that the cyclisation of this
form was faster at high and low pH values and had a mini-
mum between pH 7 and 10. These observations were used
to support parallel mechanisms involving general-base catal-
ysis and general-acid catalysis.13 In the present case, there
was a trend towards faster cyclisation at higher pH within
the range pH 6.0–8.0, which correlated with increased frac-
tions of the unprotonated reactive forms, but reactions at
more extreme pH values (outside the physiological range)
were not studied.
However, the detail of the conformation required for the
starting dipeptide amide 21 as it enters the initial nucleo-
philic reaction reveals the origin of the rate-slowing effect
of bulky R0. As can be seen in structure 21cA (Scheme 7,
derived from L-Val), the conformation in which the primary
amine can approach the target electrophile requires a partic-
ular local conformation about the carbonyl–Ca bond in
which R0 is fully eclipsing the carbonyl oxygen. Thus
increasing the steric bulk of R0 will make this conformation
increasingly unfavourable and thus slows the initial step of
the cyclisation.
In the L,S diastereomeric series, which also includes Gly-R-
DmtNH(CH2)2C6H4NO2 21a, the opposite trend in overall
cyclisation reaction rates is evident, with increasing
local steric bulk of the side-chain R0 tending to speed the
reaction (Table 1). The Gly analogue 21a may be a special
case, as noted above, involving unfavourable populations
of the conformers in equilibrium E (Scheme 7). Addition-
ally, the rates of cyclisation of the major members of this
series, L-Val-S-DmtNH(CH2)2C6H4NO2 40c and L-Leu-S-
DmtNH(CH2)2C6H4NO2 40e, are also much faster than
those of the L,R series, with the L,S Val analogue 40c reacting
ca. 100 times faster than its diastereoisomer 21c. As with the
L,R series, examination of the structures of the reacting con-
formations enabled rationalisation of the relative rates.
Structure 41cA shows the conformation of the L-Val derived
DKP, as discussed above. In this structure, the isopropyl
group is not close in space to either of the geminal dimethyl
groups, so variation of this group would not be expected to
influence the energy of the DKP greatly. This group is also
In this paper, we have reported the cyclisations of two dia-
stereomeric series of aminoacyl-Dmt N-(2-(4-nitrophenyl)-
ethyl)amides, giving the cyclic dipeptides (DKPs) and
releasing 2-(4-nitrophenyl)ethylamine as a model for drugs
carrying primary amines. The rate of ring-closure and release
depends on the relative configurations of the two chiral
centres, the L,S dipeptide amides react much more rapidly.
Each of the Aaa-Dmt dipeptides adopts 80–100% the cis ter-
tiary amide conformation; thus the rate of ring-closure is

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11257
effectively independent of cis–trans interconversion. Thus
the L-Val-L-Dmt and L-Leu-L-Dmt units are shown to be
highly effective self-immolative molecular clips for incorpo-
ration into prodrug linkers designed for cleavage/triggering
by endopeptidases. These linkers release amine drugs rap-
idly, whereas the existing dipeptide cyclisation technol-
ogy11,12 is effective only in releasing OH-containing drugs
where the ester is a better electrophile and the alcohol is a
better leaving group. Aminoacyl-Dmt units, therefore, have
great potential utility in prodrug design and development.
3.03 (d, J¼12.1 Hz) and 3.34 (m, 5-H2), 4.67 (0.6H, dd,
J¼4, 2 Hz) and 4.74 (0.4H, br d, J¼5 Hz, 4-H); NMR
((CD3)2SO, 80 ꢀC) dH 1.41 (9H, s, But), 1.73 (3H, s,
2-Me), 1.77 (3H, s, 2-Me), 3.05 (1H, dd, J¼12.1, 3.0 Hz,
5-H), 3.35 (1H, dd, J¼12.1, 6.9 Hz, 5-H), 4.72 (1H, dd,
J¼6.9, 3.0 Hz, 4-H).
8.4. Pentafluorophenyl R-3-(1,1-dimethylethoxycar-
bonyl)-2,2-dimethyltetrahydrothiazole-4-carboxylate(13)
Compound 12 (1.10 g, 4.2 mmol) was stirred with penta-
fluorophenol (850 mg, 4.6 mmol) and DCC (850 mg,
4.6 mmol) in EtOAc (10 mL) at 0 ꢀC under N2 for 3 h. Filtra-
tion and evaporation gave 13 (1.62 g, 90%) as white needles:
mp 107–109 ꢀC (lit.27 mp 104–105 ꢀC); NMR dH 1.46
(6.3H, s, But), 1.52 (2.7H, s, But), 1.80 (0.9H, s, 2-Me),
1.83 (3H, s, 2-Me), 1.90 (2.1H, s, 2-Me), 3.28 (0.7H, br d,
J¼12.5 Hz, 5-H), 3.41 (0.3H, m, 5-H), 3.47 (1H, dd, J¼
12.5, 7.0 Hz, 5-H), 4.05 (0.3H, m, 5-H), 5.18 (0.7H,
dd, J¼6.6, 1.6 Hz, 4-H), 5.26 (0.3H, dd, J¼6.6, 2.3 Hz,
4-H); NMR dF ꢁ162.2 (0.3F, m, 40-F), ꢁ161.8 (0.7F, m,
40-F), ꢁ157.8 (0.6F, t, J¼21.1 Hz, 30,50-F2), ꢁ157.3 (1.4F, t,
J¼21.1 Hz, 30,50-F2), ꢁ152.6 (1.4F, d, J¼17.2 Hz, 20,60-F2),
ꢁ151.90 (0.6F, d, J¼17.2 Hz, 20,60-F2).
8. Experimental
8.1. General
1H NMR spectra were recorded on Varian GX270 or EX400
spectrometers of samples in CDCl3, unless otherwise stated.
IR spectra were recorded on a Perkin–Elmer 782 spectrom-
eter as KBr discs, unless otherwise stated. Mass spectra were
obtained using fast atom bombardment (FAB) ionisation in
the positiveion mode, unless otherwisestated. The chromato-
graphic stationary phase was silica gel. DCC refers to N,N0-
dicyclohexylcarbodiimide, THF refers to tetrahydrofuran,
DMFreferstodimethylformamide, HOBtrefersto1-hydroxy-
benzotriazole, DMAP refers to 4-dimethylaminopyridine and
citric acid refers to a 5% aqueous solution of 3-carboxy-3-
hydroxypentanedioic acid. THF was dried with Na. Solutions
in organic solvents 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 either with a Thermo Galen Kofler block
(uncorrected) or with a differential scanning calorimeter.
8.5. R-2,2-Dimethyl-3-(1,1-dimethylethoxycarbonyl)-
N-(2-(4-nitrophenyl)ethyl)tetrahydrothiazole-4-
carboxamide (14)
2-(4-Nitrophenyl)ethylamine hydrochloride 24$HCl (85 mg,
0.42 mmol) was stirred with Et3N (85 mg, 0.84 mmol) and
13 (180 mg, 0.42 mmol) in CH2Cl2 (2.0 mL) for 2 h. Evapo-
ration and chromatography (CHCl3/MeOH, 9:1) afforded 14
(160 mg, 93%) as a viscous yellow oil: IR (film) nmax 3325,
1681, 1519, 1346 cmꢁ1; NMR dH 1.43 (9H, s, But), 1.70
(3H, s, 2-Me), 1.75 (3H, s, 2-Me), 2.95 (2H, m, ArCH2),
3.23 (2H, m, 5-H2), 3.53 (1H, m) and 3.68 (1H, m,
NHCH2), 4.74 (1H, m, 4-H), 7.42 (2H, d, J¼8.6 Hz, Ar
2,6-H2), 8.18 (2H, d, J¼8.6 Hz, Ar 3,5-H2); NMR dC 19.6,
28.7, 36.0, 40.7, 67.5, 82.0, 93.7, 110.0, 124.0, 129.9,
146.9, 171.3; MS m/z 410.1764 (M+H) (C19H28N3O5S re-
quires 410.1750), 354 (MꢁMe2C]CH2), 336 (MꢁButO),
310 (MꢁBoc). Found: H, 6.59; N, 9.94. C19H27N3O5S
requires H, 6.65; N, 10.27%.
8.2. R-2,2-Dimethyltetrahydrothiazole-4-carboxylic
acid hydrochloride (11)
L-Cys$HCl$H2O 10 (3.5 g, 20 mmol) was boiled under
reflux in acetone (250 mL) and 2,2-dimethoxypropane
(50 mL) for 6 h under N2. The solid was collected by filtra-
tion to afford 11 (3.45 g, 87%) as a white solid: mp 125–
130 ꢀC (lit.27 mp 165–168 ꢀC); IR nmax 3412, 2600,
1744 cmꢁ1; NMR (D2O) dH 2.06 (6H, s, 2ꢂMe), 2.92 (1H,
dd, J¼15.2, 3.9 Hz, 5-H), 3.00 (1H, dd, J¼15.2, 5.9 Hz,
5-H), 4.06 (1H, dd, J¼5.9, 3.9 Hz, 4-H).
8.6. R-2,2-Dimethyl-N-(2-(4-nitrophenyl)ethyl)tetra-
hydrothiazole-4-carboxamide trifluoroacetate salt (15)
8.3. R-3-(1,1-Dimethylethoxycarbonyl)-2,2-dimethyl-
tetrahydrothiazole-4-carboxylic acid (12)
Compound 14 (1.30 g, 3.2 mmol) was stirred with CF3CO2H
(20 mL) and CH2Cl2 (10 mL) for 2 h. Evaporation afforded
15 (1.35 g, quant.) as a highly hygroscopic gum: NMR dH
1.82 (3H, s, 2-Me), 1.93 (3H, s, 2-Me), 2.97 (2H, m,
ArCH2), 3.20 (1H, dd, J¼12.5, 6.2 Hz, 5-H), 3.59 (1H, dd,
J¼12.5, 8.2 Hz, 5-H), 3.64 (2H, m, NHCH2), 5.22 (1H, dd,
J¼8.2, 6.2 Hz, 4-H), 7.35 (2H, d, J¼8.6 Hz, Ar 2,6-H2),
7.56 (1H, br t, J¼5.9 Hz, NH), 8.15 (2H, d, J¼8.6 Hz, Ar
3,5-H2), 9.76 (2H, br, N+H2); MS m/z 310.1238 (M+H)
(C14H20N3O3S requires 310.1225).
Compound 11 (1.0 g, 5.1 mmol) was stirred with Pri2NEt
(722 mg, 5.6 mmol) and di-tert-butyl dicarbonate (1.45 g,
6.6 mmol) in dry MeCN (10 mL) for 2 days. The evaporated
residue, in Et2O, was filtered (CeliteÒ). The evaporated resi-
due, in CH2Cl2, was washed with aq H2SO4 (100 mM, cold),
H2O and brine. Drying, evaporation and recrystallisation
(hexane) afforded 12 (150 mg, 11%) as a white solid: mp
112–114 ꢀC (lit.27 mp 114–114.5 ꢀC); NMR (CDCl3,
20 ꢀC) dH 1.43 (4.5H, br s) and 1.53 (4.5H, br s, But), 1.80
(4.5H, m) and 1.87 (1.5H, br s, 2ꢂMe), 3.1–3.28 (2H, m,
5-H2), 4.83 (1H, m) and 4.98 (1H, m, 4-H); NMR
((CD3)2SO, 20 ꢀC) dH 1.35 (5.4H, br s) and 1.43 (3.6H, br
s) (But), 1.70 (br s), 1.73 (br s) and 1.75 (br s, 2ꢂMe),
8.7. 1,1-Dimethylethyl N-(fluorocarbonylmethyl)carba-
mate (17a)
BocGlyOH 16a (1.0 g, 5.7 mmol) was stirred with pyridine
(450 mg, 5.7 mmol) in dry CH2Cl2 (30 mL) and added

11258
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
slowly to 2,4,6-trifluoro-1,3,5-triazine (1.54 g, 11.4 mmol)
in dry CH2Cl2 (10 mL), then stirred for 3 h. The mixture
was washed with ice-water (3ꢂ). Drying and evaporation
afforded crude 17a (800 mg) as a white gum: NMR dH
1.47 (9H, s, But), 4.11 (2H, t, J¼5.5 Hz, CH2), 5.05 (1H,
br, NH); NMR dF 30.21 (1F, s, CO2F).
18b (30% from 11) as a pale yellow solid: mp 93–94 ꢀC:
IR nmax 3338, 1705, 1655, 1534 cmꢁ1; NMR dH 1.31 (3H,
d, J¼6.6 Hz, Ala-Me), 1.40 (9H, s, But), 1.81 (3H, s,
2-Me), 1.92 (3H, s, 2-Me), 3.24 (1H, dd, J¼12.1, 5.5 Hz,
5-H), 3.45 (1H, d, J¼12.1 Hz, 5-H), 4.53 (1H, qn, J¼7 Hz,
Ala a-H), 4.85 (1H, d, J¼5.1 Hz, 4-H), 5.68 (1H, d,
J¼7.8 Hz, NH); MS m/z 333.1489 (M+H) (C14H25N2O5S
requires 333.1484), 277 (MꢁMe2C]CH2), 259 (MꢁButO).
8.8. 1,1-Dimethylethyl S-N-(1-fluorocarbonyl-2-methyl-
propyl)carbamate (17c)
8.12. R-2,2-Dimethyl-3-(N-(1,1-dimethylethoxy-
carbonyl)-L-valinyl)tetrahydrothiazole-4-carboxylic
acid (18c)
BocValOH 16c (2.5 g, 11 mmol) was stirred with pyridine
(910 mg, 11.5 mmol) in dry CH2Cl2 (30 mL) and added
slowly to 2,4,6-trifluoro-1,3,5-triazine (3.1 g, 23 mmol) in
dry CH2Cl2 (30 mL). Stirring was continued under N2 at
ꢁ10 ꢀC for 2 h. The mixture was washed with ice-water
(3ꢂ100 mL). Drying and evaporation afforded crude 17c
(2.22 g) as a white solid: mp 38–42 ꢀC (lit.39 mp 36–
38 ꢀC); NMR dH 0.99 (3H, d, J¼7.0 Hz, Val-Me), 1.04
(3H, d, J¼6.6 Hz, Val-Me), 1.46 (9H, s, But), 2.25 (1H, m,
Val b-H), 4.40 (1H, dd, J¼8.6, 4.7 Hz, Val a-H), 4.96 (1H,
br d, NH); NMR dF 32.29 (1F, s, CO2F).
Compound 17c was treated with 11, as for the synthesis of
18a except that the chromatographic eluant was hexane/
EtOAc/AcOH (70:29:1), to give 18c (27%) as a colourless
gummy solid: IR nmax 3338, 1705, 1655, 1534 cmꢁ1
;
NMR dH 0.90 (3H, d, J¼7.0 Hz, Val-Me), 1.0 (3H, d,
J¼6.6 Hz, Val-Me), 1.39 (9H, s, But), 1.82 (3H, s, 2-Me),
1.88 (1H, m, Val b-H), 1.92 (3H, s, 2-Me), 3.21 (1H, dd,
J¼12.1, 5.5 Hz, 5-H), 3.44 (1H, d, J¼12.1 Hz, 5-H), 4.50
(1H, dd, J¼9.4, 5.5 Hz, Val a-H), 5.02 (1H, d, J¼5.1 Hz,
4-H), 5.60 (1H, d, J¼9.4 Hz, NH), 8.52 (1H, br, OH); MS
m/z 361.1822 (M+H) (C16H29N2O5S requires 361.1797),
305 (MꢁMe2C]CH2), 261 (MꢁBoc).
8.9. 1,1-Dimethylethyl S-N-(1-fluorocarbonyl-2-phenyl-
ethyl)carbamate (17d)
BocPheOH 16d was treated with pyridine and 2,4,6-tri-
fluoro-1,3,5-triazine, as for the synthesis of 17c, to give
crude 17d (57%) as a colourless oil: NMR dH 1.42 (9H, s,
But), 3.15 (1H, dd, J¼14.4, 6.2 Hz, Phe b-H), 3.17 (1H,
dd, J¼14.4, 5.9 Hz, Phe b-H), 4.74 (1H, br d, J¼5.9 Hz,
Phe a-H), 4.86 (1H, d, J¼6.6 Hz, NH), 7.10–7.30 (5H, m,
Ph-H5); NMR dF 29.21 (1F, s, CO2F).
8.13. R-2,2-Dimethyl-3-(N-(1,1-dimethylethoxy-
carbonyl)-L-phenylalanyl)tetrahydrothiazole-
4-carboxylic acid (18d)
Compound 17d was treated with 11, as for the synthesis of
18a except that the chromatographic eluant was EtOAc/
AcOH (99:1), to give 18d (30%) as a pale yellow gum:
NMR dH 1.29 (9H, s, But), 1.68 (3H, s, 2-Me), 1.77 (3H, s,
2-Me), 2.80 (1H, dd, J¼13.3, 7.1 Hz, Phe b-H), 2.88 (1H,
dd, J¼13.3, 6.1 Hz, Phe b-H), 2.97 (1H, dd, J¼11.7,
5.9 Hz, 5-H), 3.12 (1H, d, J¼11.7 Hz, 5-H), 4.34 (1H, ddd,
J¼9.0, 7.1, 6.1 Hz, Phe a-H), 4.68 (1H, d, J¼5.9 Hz, 4-H),
6.50 (1H, d, J¼9.0 Hz, NH), 7.26 (5H, m, Ph-H5); MS m/z
409.1812 (M+H) (C20H29N2O5S requires 409.1797), 353
(MꢁMe2C]CH2), 335 (MꢁButO), 309 (MꢁBoc).
8.10. R-2,2-Dimethyl-3-(N-(1,1-dimethylethoxy-
carbonyl)glycyl)-2,2-dimethyltetrahydrothiazole-
4-carboxylic acid (18a)
Compound 17a (810 mg, 4.6 mmol) was stirred with 11
(1.00 g, 5.1 mmol) and Pr2i NEt (1.24 g, 9.6 mmol) in dry
DMF (100 mL) for 3 h. The evaporated residue, in EtOAc,
was washed with cold citric acid and brine. Drying, evapo-
ration and chromatography (EtOAc/Et2O/AcOH, 13:6:1)
afforded 18a (560 mg, 35%) as a pale buff solid: mp 152–
155 ꢀC: IR nmax 3385, 1748, 1672, 1625, 1539 cmꢁ1
;
8.14. Pentafluorophenyl R-2,2-dimethyl-3-(N-(1,1-
dimethylethoxycarbonyl)glycyl)tetrahydrothiazole-
4-carboxylate (19a)
NMR ((CD3)2SO) dH 1.38 (9H, s, But), 1.74 (3H, s, 2-Me),
1.76 (3H, s, 2-Me), 3.28 (2H, m, 5-H2), 3.50 (1H, dd,
J¼17.2, 6.2 Hz) and 3.87 (1H, dd, J¼16.8, 5.5 Hz,
Gly-H2), 5.10 (1H, m, 4-H), 6.78 (1H, t, J¼6 Hz, NH);
MS m/z 319.1339 (M+H) (C13H23N2O5S requires
319.1328), 263 (MꢁMe2C]CH2). Found: C, 47.80;
H, 6.72; N, 8.85. C13H22N2O5S$0.5H2O requires C, 47.99;
H, 6.51; N, 8.61%.
Compound 18a (450 mg, 1.4 mmol) was stirred with penta-
fluorophenol (290 mg, 1.6 mmol) and DCC (330 mg,
1.6 mmol) in EtOAc (5.0 mL) at 0 ꢀC under N2 for 3 h.
The mixture was filtered (CeliteÒ) and the solid was washed
with cold EtOAc. The evaporated residue, in hexane, was
kept at 4 ꢀC for 16 h and filtered. Evaporation gave 19a
(700 mg, 92%) as a colourless oil: NMR dH 1.45 (9H, s,
But), 1.88 (3H, s, 2-Me), 1.92 (3H, s, 2-Me), 3.45 (1H, dd,
J¼12.5, 5.5 Hz, 5-H), 3.51 (1H, d, J¼12.1 Hz, 5-H), 3.67
(1H, dd, J¼17.2, 3 Hz) and 4.13 (1H, dd, J¼17.2, 6.6 Hz)
(Gly-H2), 5.20 (1H, d, J¼5.3 Hz, 4-H), 5.37 (1H, br,
NH); NMR dF ꢁ161.3 (2F, m, 30,50-F2), ꢁ156.4 (1F,
t, J¼21.4 Hz, 40-F), ꢁ152.0 (2F, d, J¼16.8 Hz, 20,60-F2);
MS m/z 485.1185 (M+H) (C19H22F5N2O5S requires
485.1170), 429 (MꢁMe2C]CH2), 411 (MꢁButO), 385
(MꢁBoc).
8.11. 1,1-Dimethylethyl S-N-(1-(fluorocarbonyl)ethyl)-
carbamate (17b) and R-2,2-dimethyl-3-(N-(1,1-
dimethylethoxycarbonyl)-L-alanyl)tetrahydrothiazole-
4-carboxylic acid (18b)
BocAlaOH 16b was treated with pyridine and 2,4,6-tri-
fluoro-1,3,5-triazine, as for the synthesis of 17c, to give
crude 17b as a white solid. This material was treated with
11, as for the synthesis of 18a except that the chromato-
graphic eluant was EtOAc/Et2O/AcOH (49:49:2), to give

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11259
8.15. Pentafluorophenyl R-2,2-dimethyl-3-(N-(1,1-
dimethylethoxycarbonyl)-L-alanyl)tetrahydrothiazole-
4-carboxylate (19b)
dF ꢁ161.8 (2F, dd, J¼21.0, 17.1 Hz, 30,50-F2), ꢁ156.9
(1F, t, J¼21.0 Hz, 40-F), ꢁ150.7 (1F, d, J¼18.4 Hz, 20,60-
F2); MS m/z 575.1635 (M+H) (C26H28F5N2O5S requires
575.1639), 519 (MꢁMe2C]CH2), 501 (MꢁButO), 475
(MꢁBoc).
Compound 18b was treated with pentafluorophenol and
DCC, as for the synthesis of 19a, to give 19b (89%) as a
colourless oil: NMR dH 1.36 (2.1H, d, J¼6.6 Hz, Ala-Me),
1.37 (0.9H, d, J¼6.6 Hz, Ala b-H3), 1.39 (6.3H, s, But),
1.43 (2.7H, s, But), 1.85 (2.1H, s, 2-Me), 1.94 (2.1H, s,
2-Me), 2.05 (0.9H, s, 2-Me), 2.09 (0.9H, s, 2-Me), 3.33
(0.3H, dd, J¼12.7, 2.9 Hz, 5-H), 3.40 (0.7H, dd, J¼12.5,
5.5 Hz, 5-H), 3.46 (0.3H, m, 4-H), 3.49 (0.7H, d,
J¼12.1 Hz, 4-H), 4.39 (0.7H, qn, J¼7 Hz, Ala a-H), 4.79
(0.3H, qn, J¼7 Hz, Ala a-H), 5.19 (0.7H, d, J¼5.5 Hz,
4-H), 5.24 (0.3H, br, NH), 5.40 (0.7H, d, J¼7.8 Hz, NH),
5.57 (0.3H, dd, J¼6.6, 3.1 Hz, 4-H); NMR dF ꢁ161.7
(0.6F, m, 30,50-F2), ꢁ161.6 (1.4F, dt, J¼21.0, 18.4 Hz,
30,50-F2), ꢁ157.1 (0.3F, t, J¼21.0 Hz, 40-F), ꢁ156.7 (0.7F,
t, J¼21.0 Hz, 40-F), ꢁ151.9 (0.6F, d, J¼17.1 Hz, 20,60-F2),
ꢁ150.8 (1.4F, d, J¼17.1 Hz, 20,60-F2); MS m/z 499.1335
(M+H) (C20H24F5N2O5S requires 499.1326), 443
(MꢁMe2C]CH2), 425 (MꢁButO).
8.18. R-2,2-Dimethyl-3-(N-(1,1-dimethylethoxycarbo-
nyl)glycyl)-N-(2-(4-nitrophenyl)ethyl)tetrahydrothia-
zole-4-carboxamide (20a)
Compound 19a was treated with 24$HCl and Et3N, as for the
synthesis of 14 except that the chromatographic eluant was
EtOAc, to give 20a (76%) as a white solid: mp 65–66 ꢀC;
NMR ((CD3)2SO, 80 ꢀC) dH 1.38 (9H, s, But), 1.72 (3H, s,
2-Me), 1.74 (3H, s, 2-Me), 2.91 (2H, t, J¼7.4 Hz, ArCH2),
3.10 (1H, d, J¼12.9 Hz, 5a-H), 3.20 (1H, dd, J¼16.4,
5.5 Hz, Gly-H), 3.31 (1H, m, 5b-H), 3.39 (1H, m) and 3.47
(1H, m) (ArCH2CH2), 3.67 (1H, dd, J¼16.4, 5.9 Hz, Gly-
H), 4.79 (1H, d, J¼5.5 Hz, 4-H), 6.67 (1H, br t, NH), 7.50
(2H, d, J¼8.6 Hz, Ar 2,6-H2), 8.13 (2H, d, J¼8.6 Hz, Ar
3,5-H2); MS m/z 467.1943 (M+H) (C21H31N4O6S requires
467.1964), 411 (MꢁMe2C]CH2), 393 (MꢁButO), 367
(MꢁBoc).
8.16. Pentafluorophenyl 2,2-dimethyl-3-(N-(1,1-
dimethylethoxycarbonyl)-L-valinyl)tetrahydrothiazole-
4-carboxylate (19c)
8.19. R-2,2-Dimethyl-3-(N-(1,1-dimethylethoxycar-
bonyl)-L-alanyl)-N-(2-(4-nitrophenyl)ethyl)tetrahydro-
thiazole-4-carboxamide (20b)
Compound 18c was treated with pentafluorophenol and
DCC, as for the synthesis of 19a, to give 19c (93%) as a col-
ourless oil: IR (film) nmax 3440, 1718, 1669, 1521 cmꢁ1
;
Compound 19b was treated with 24$HCl and Et3N, as for the
synthesis of 14 except that the chromatographic eluant was
EtOAc/hexane (4:1), to give 20b (75%) as a white solid:
mp 156–158 ꢀC: IR nmax 3319, 1696, 1657, 1601,
1520 cmꢁ1; NMR ((CD3)2SO) dH 1.12 (3H, d, J¼7.0 Hz,
Ala-Me), 1.37 (9H, s, But), 1.61 (3H, s, 2-Me), 1.71 (3H,
s, 2-Me), 2.95 (2H, m, ArCH2), 3.24 (1H, dd, J¼12.1,
5.5 Hz, 5b-H), 3.32 (2H, m, 5a-H+ArCH2CH), 3.53 (1H,
m, ArCH2CH), 3.84 (1H, m, Ala a-H), 4.73 (1H, d,
J¼5.5 Hz, 4-H), 7.06 (1H, d, J¼5.9 Hz, NH), 7.49 (2H, d,
J¼9.0 Hz, Ar 2,6-H2), 8.13 (2H, d, J¼8.6 Hz, Ar 3,5-H2)
8.13 (1H, br, NH); MS m/z 481.2142 (M+H)
(C22H33N4O6S requires 481.2121), 425 (MꢁMe2C]CH2),
381 (MꢁBoc).
NMR dH 0.94 (2.1H, d, J¼6.6 Hz, Val-Me), 0.99 (2.1H, d,
J¼6.6 Hz, Val-Me), 1.03 (0.9H, d, J¼7.0 Hz, Val-Me),
1.10 (0.9H, d, J¼7.0 Hz, Val-Me), 1.33 (6.3H, s, But), 1.43
(2.7H, s, But), 1.86 (2.1H, s, 2-Me), 1.91 (0.7H, m, Val
b-H), 1.95 (2.1H, s, 2-Me), 1.99 (1.8H, s, 2ꢂ2-Me),
3.32 (0.3H, dd, J¼12.5, 3.5 Hz, 5-H), 3.40 (0.7H, dd,
J¼12.5, 5.5 Hz, 5-H), 3.44 (0.3H, d, J¼6.2 Hz, 5-H),
3.50 (0.7H, d, J¼12.1 Hz, 5-H), 4.28 (0.7H, dd, J¼9.0,
6.2 Hz, Val a-H), 4.54 (0.3H, dd, J¼9.8, 6.2 Hz, Val a-H),
5.12 (0.3H, d, J¼10.2 Hz, NH), 5.24 (0.7H, d, J¼9.0 Hz,
NH), 5.38 (0.7H, d, J¼4.7 Hz, 4-H), 5.62 (0.3H,
dd, J¼6.6, 3.5 Hz, 4-H); NMR dF ꢁ161.9 (1.4F, dd,
J¼22.4, 18.4 Hz, 30,50-F2), ꢁ161.6 (0.6F, dd, J¼21.0,
17.1 Hz, 30,50-F2), ꢁ157.1 (0.7F, dt, J¼22.4 Hz, 40-F),
ꢁ152.6 (0.3F, dt, J¼17.1 Hz, 40-F), ꢁ151.8 (0.6F,
d, J¼17.1 Hz, 20,60-F2), ꢁ150.3 (1.4F, d, J¼18.4 Hz,
20,60-F2); MS m/z 527.1660 (M+H) (C22H28F5N2O5S re-
quires 527.1639), 471 (MꢁButO), 427 (MꢁBoc), 328
(MꢁBocVal).
8.20. R-(2,2-Dimethyl-3-(1,1-dimethylethoxycarbonyl)-
L-valinyl)-N-(2-(4-nitrophenyl)ethyl)tetrahydrothia-
zole-4-carboxamide (20c)
Compound 19c was treated with 24$HCl and Et3N, as for the
synthesis of 14 except that the chromatographic eluant was
EtOAc/hexane (1:1), to give 20c (73%) as a white solid:
mp 233.8 ꢀC (DSC); IR nmax 3331, 1700, 1653,
1519 cmꢁ1; NMR dH 0.95 (3H, d, J¼6.6 Hz, Val-Me),
1.01 (3H, d, J¼6.6 Hz, Val-Me), 1.44 (9H, s, But), 1.65
(3H, s, 2-Me), 1.81 (3H, s, 2-Me), 1.87 (1H, m, Val b-H),
3.02 (2H, m, ArCH2), 3.12 (1H, dd, J¼12.1, 5.5 Hz, 5b-
H), 3.50 (1H, m, ArCH2CH), 3.63 (1H, d, J¼12.1 Hz, 5a-
H), 3.75 (1H, m, Val a-H), 3.79 (1H, m, ArCH2CH), 4.67
(1H, d, J¼5.5 Hz, 4-H), 4.90 (1H, d, J¼7.8 Hz, NHBoc),
7.38 (2H, d, J¼9.0 Hz, Ar 2,6-H2), 7.98 (1H, br, NHCH2),
8.14 (2H, d, J¼8.6 Hz, Ar 3,5-H2); MS m/z 509.2434
(M+H) (C24H37N4O6S requires 509.2434), 453 (MꢁButO),
409 (MꢁBoc), 310 (MꢁBocVal).
8.17. Pentafluorophenyl R-2,2-dimethyl-3-(N-(1,1-
dimethylethoxycarbonyl)-L-phenylalanyl)tetra-
hydrothiazole-4-carboxylate (19d)
Compound 18d was treated with pentafluorophenol and
DCC, as for the synthesis of 19a, to give 19d (92%) as
a white solid: mp 35-36 ꢀC; IR nmax 3442, 1792, 1716,
1659, 1523 cmꢁ1; NMR dH 1.39 (9H, s, But), 1.77 (3H, s,
2-Me), 1.86 (3H, s, 2-Me), 2.40 (1H, dd, J¼12.5, 5.5 Hz,
5-H), 2.89 (1H, dd, J¼12.5, 10.9 Hz, Phe b-H), 3.00 (1H,
d, J¼12.5 Hz, 5-H), 3.25 (1H, dd, 12.5, 3.9 Hz, Phe b-H),
4.37 (1H, d, J¼5.5 Hz, 4-H), 4.51 (1H, m, Phe a-H),
5.42 (1H, d, J¼7.8 Hz, NH), 7.33 (5H, m, Ph-H5); NMR

11260
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
8.21. R-2,2-Dimethyl-3-(N-(1,1-dimethylethoxycar-
bonyl)-L-phenylalanyl)-N-(2-(4-nitrophenyl)ethyl)-
tetrahydrothiazole-4-carboxamide (20d)
3.45 (1H, dd, J¼12.1, 5.9 Hz, 5-H), 3.53 (3H, m, NCH2
and Val a-H), 4.95 (1H, dd, J¼5.9, 2.3 Hz, 4-H), 7.57 (2H,
d, J¼8.6 Hz, Ar 2,6-H2), 8.16 (3H, br, N+H3), 8.23 (2H,
d, J¼8.6 Hz, Ar 3,5-H2), 8.33 (1H, t, J¼5.9 Hz, NH); MS
m/z 409.1911 (M+H) (C19H29N4O4S requires 409.1910).
Compound 19d was treated with 24$HCl and Et3N, as for the
synthesis of 20c, to give 20d (95%) as a white solid: mp 45–
46 ꢀC: IR nmax 3442, 1699, 1652, 1518 cmꢁ1; NMR dH 1.43
(9H, s, But), 1.58 (3H, s, 2-Me), 1.60 (3H, s, 2-Me), 2.30
(1H, dd, J¼11.7, 5.9 Hz, Phe b-H), 2.89 (2H, m, Phe
b-H+5b-H), 2.97 (2H, t, J¼7.0 Hz, ArCH2), 3.20 (1H, d,
J¼11.7 Hz, 5a-H), 3.41 (1H, m, ArCH2CH), 3.69 (1H, m,
ArCH2CH), 3.74 (1H, d, J¼5.5 Hz, 4-H), 4.13 (1H, m, Phe
a-H), 4.98 (1H, d, J¼5.9 Hz, Phe NH), 7.18 (2H, m, Phe
20,60-H2), 7.33 (3H, m, Phe 30,40,50-H3), 7.35 (2H, d, J¼
8.6 Hz, Ar 2,6-H2), 8.10 (2H, d, J¼9 Hz, Ar 3,5-H2), 8.10
(1H, m, NH); MS m/z 557.2433 (M+H) (C28H37N4O6S
requires 557.2434), 501 (MꢁMe2C]CH2), 457 (MꢁBoc).
8.25. R-2,2-Dimethyl-N-(2-(4-nitrophenyl)ethyl)-3-
(L-phenylalanyl)tetrahydrothiazole-4-carboxamide
trifluoroacetate salt (21d)
Compound 20d was treated with CF3CO2H, as for the syn-
thesis of 21c, to give 21d (quant.) as a colourless highly
hygroscopic gum: NMR (CD3OD) dH 1.64 (3H, s, 2-Me),
1.70 (3H, s, 2-Me), 2.55 (1H, dd, J¼12.5, 5.9 Hz, 5-H),
2.90 (2H, t, J¼7.0 Hz, ArCH2), 2.95 (1H, dd, J¼13.3,
8.6 Hz, Phe b-H), 3.08 (1H, d, J¼12.5 Hz, 5-H), 3.10 (1H,
dd, J¼13.3, 5.6 Hz, Phe b-H), 3.42 (2H, m, NHCH2), 4.08
(1H, m, Phe a-H), 4.20 (1H, d, J¼4.6 Hz, 4-H), 4.98 (1H,
d, J¼5.9 Hz, Phe NH), 7.27 (2H, m, Phe 20,60-H2), 7.36
(3H, m, Phe 30,40,50-H3), 7.50 (2H, d, J¼8.6 Hz, Ar
2,6-H2), 8.18 (2H, d, J¼8.6 Hz, Ar 3,5-H2), 8.23 (1H, t,
J¼6.6 Hz, NH), 8.38 (3H, br, N+H3); MS m/z 457.1907
(M+H) (C23H29N4O4S requires 457.1910).
8.22. R-2,2-Dimethyl-3-glycyl-N-(2-(4-nitrophenyl)-
ethyl)tetrahydrothiazole-4-carboxamide trifluoro-
acetate salt (21a)
Compound 20a (50 mg, 0.11 mmol) was stirred in
CF3CO2H (0.4 mL) and CH2Cl2 (1.6 mL) for 45 min. Evap-
oration afforded 21a (53 mg, quant.) as a highly hygroscopic
colourless gum: NMR ((CD3)2SO) dH 1.76 (3H, s, 2-Me),
1.78 (3H, s, 2-Me), 2.90 (2H, t, J¼6.6 Hz, ArCH2), 3.18
(1H, m, Gly-H), 3.20 (d, J¼12.1 Hz, 5-H), 3.34 (1H, dd,
J¼12.5, 5.9 Hz, 5-H), 3.42 (2H, br q, ArCH2CH2), 3.89
(1H, m, Gly-H), 4.85 (1H, d, J¼6.2 Hz, 4-H), 7.50 (2H, d,
J¼9.0 Hz, Ar 2,6-H2), 8.02 (3H, br, N+H3), 8.15 (2H,
d, J¼8.6 Hz, Ar 3,5-H2), 8.26 (1H, t, J¼6.2 Hz, NH); MS
m/z 367.1448 (M+H) (C16H24N4O4S requires 367.1400),
310 (MꢁGly), 225 (MꢁCH2CH2C6H4NO2).
8.26. Pentafluorophenyl 3-(L-alanyl)-R-2,2-dimethyl-
tetrahydrothiazole-4-carboxylate hydrochloride (22b)
HCl was bubbled through 19b (90 mg, 0.18 mmol) in
CH2Cl2 (5.0 mL) for 30 min. Evaporation afforded 22b
(quant.) as a highly hygroscopic yellow solid: IR nmax
3423, 1793, 1670, 1521 cmꢁ1; NMR ((CD3)2SO) dH 1.20
(3H, d, J¼7.0 Hz, Ala-Me), 1.74 (3H, s, 2-Me), 1.77 (3H,
s, 2-Me), 3.17 (1H, dd, J¼12.1, 10.2 Hz, 5-H), 3.25 (1H,
dd, J¼12.1, 6.6 Hz, 5-H), 4.10 (1H, q, J¼7.0 Hz, Ala
a-H), 4.68 (1H, dd, J¼10.4, 6.6 Hz, 4-H), 6.30 (3H, br,
N+H3); MS m/z 215 (MꢁC6F5O).
8.23. 3-(L-Alanyl)-R-2,2-dimethyl-N-(2-(4-nitrophenyl)-
ethyl)tetrahydrothiazole-4-carboxamide trifluoro-
acetate salt (21b)
8.27. Pentafluorophenyl R-2,2-dimethyl-3-(L-valinyl)-
tetrahydrothiazole-2-carboxylate hydrochloride (22c)
Compound 20b was treated with CF3CO2H, as for the syn-
thesis of 21a except that the reaction time was 20 min, to
give 21b (quant.) as a pale buff solid: mp 119–121 ꢀC: IR
nmax 3410, 1655, 1600, 1517 cmꢁ1; NMR (CD3OD) dH
1.34 (3H, d, J¼6.6 Hz, Ala-Me), 1.69 (3H, s, 2-Me), 1.72
(3H, s, 2-Me), 2.85 (1H, dt, J¼12.9, 6.6 Hz) and 2.89 (1H,
dt, J¼12.9, 6.6 Hz, ArCH2), 3.10 (1H, dd, J¼12.9, 1.6 Hz,
5-H), 3.33 (2H, m, 5-H, NCHH), 3.54–3.61 (1H, dt,
J¼13.7, 6.6 Hz, NCHH), 3.68 (1H, q, J¼7.0 Hz, Ala a-H),
4.74 (1H, dd, J¼5.9, 1.6 Hz, 4-H), 7.39 (2H, d, J¼9.0 Hz,
Ar 2,6-H2), 8.07 (2H, d, J¼9.0 Hz, Ar 3,5-H2); MS m/z
381.1603 (M+H) (C17H25N4O4S requires 381.1597).
Compound 19c was treated with HCl, as for the synthesis of
22b except that the reaction time was 1 h, to give 22c
(120 mg, 90%) as a white wax: IR nmax 3433, 1790, 1667,
1520 cmꢁ1; NMR ((CD3)2SO) dH 0.82 (3H, d, J¼6.6 Hz,
Val-Me), 0.99 (3H, d, J¼7.4 Hz, Val-Me), 1.75 (6H, s,
2ꢂMe), 2.29–2.37 (1H, d septet, J¼2.3, 7.0 Hz, Val b-H),
3.13 (1H, dd, J¼11.3, 10.2 Hz, 5b-H), 3.22 (1H, dd,
J¼11.7, 6.2 Hz, 5a-H), 3.89 (1H, m, Val a-H), 4.60–4.64
(1H, dd, J¼6.2, 1.2 Hz, 4-H), 5.94 (3H, br, N+H3); NMR
dF ꢁ171.5 (1F, tt, J¼23.7, 6.8 Hz, 40-F2), ꢁ165.1 (2F, dd,
J¼23.7, 19.6 Hz, 30,50-F2), ꢁ161.5 (2F, dd, J¼19.6,
6.8 Hz, 20,60-F2); MS m/z 427 (M+H), 243 (MꢁC6F5O).
8.24. R-2,2-Dimethyl-N-(2-(4-nitrophenyl)ethyl)-3-
(L-valinyl)tetrahydrothiazole-4-carboxamide
trifluoroacetate salt (21c)
8.28. Pentafluorophenyl R-2,2-dimethyl-3-(L-phenyl-
alanyl)tetrahydrothiazole-2-carboxylate hydrochloride
(22d)
Compound 20c was treated with CF3CO2H, as for the syn-
thesis of 21a except that the reaction time was 15 min, to
give 21c (quant.) as a colourless highly hygroscopic gum:
NMR ((CD3)2SO) dH 0.95 (3H, d, J¼6.6 Hz, Val-Me),
1.00 (3H, d, J¼6.6 Hz, Val-Me), 1.80 (3H, s, 2-Me), 1.82
(3H, s, 2-Me), 2.10 (1H, m, Val b-H), 2.98 (2H, t,
J¼7.0 Hz, ArCH2), 3.32 (1H, dd, J¼12.5, 2.3 Hz, 5-H),
Compound 19d was treated with HCl, as for the synthesis of
22b except that the reaction time was 1.5 h, to give 22d
(quant.) as a colourless highly hygroscopic viscous oil: IR
nmax 3434, 1791, 1669, 1520 cmꢁ1; NMR ((CD3)2SO) dH
1.66 (3H, s, 2-Me), 1.74 (3H, s, 2-Me), 2.75 (1H, t, J¼
10.9 Hz, 5b-H), 3.04 (1H, dd, J¼14.1, 4.7 Hz, Phe b-H),

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11261
3.06 (1H, dd, J¼14.1, 4.3 Hz, Phe b-H), 3.13 (1H, dd,
J¼11.7, 5.9 Hz, 5a-H), 4.34 (1H, t, J¼4.3 Hz, Phe a-H),
4.46 (3H, br, N+H3), 4.60 (1H, dd, J¼10.5, 5.5 Hz, 4-H),
7.25 (5H, m, Ph-H5), 8.31 (1H, s, NH); NMR dF ꢁ171.5
(2F, tt, J¼22.4, 6.6 Hz, 40-F2), ꢁ165.1 (1F, dd, J¼22.4,
19.7 Hz, 30,50-F2), ꢁ161.8 (2F, dd, J¼19.7, 6.6 Hz, 20,60-
F2), MS m/z 475.1131 (M+H) (C21H20F5N2O3S requires
475.1115), 291 (MꢁC6F5O).
2.5 mmol) and Et3N (480 mg, 4.8 mmol) in dry DMF
(50 mL) for 16 h. The evaporated residue, in EtOAc, was
washed with cold citric acid and brine. Drying and evapora-
tion afforded a mixture of 27 and 18b (300 mg, 36%) as
a white solid: mp 153–155 and 160–162 ꢀC; IR nmax 3316,
1748, 1718, 1643, 1660, 1540, 1512 cmꢁ1; NMR dH 1.30
(1.2H, d, J¼2.7 Hz, Ala-Me (18b)), 1.32 (1.8H, d,
J¼3.1 Hz, Ala-Me (27)), 1.40 (3.6H, s, But (18b)), 1.42
(5.4H, s, But (27)), 1.81 (1.2H, s, 2-Me (18b)), 1.84 (1.8H,
s, 2-Me (27)), 1.87 (1.8H, s, 2-Me (27)), 1.91 (1.2H, s,
2-Me (18b)), 3.23 (0.4H, dd, J¼11.7, 5.5 Hz, 5-H (18b)),
3.34 (0.6H, dd, J¼12.1, 5.5 Hz, 5-H (27)), 3.40 (0.6H, d,
J¼12.1 Hz, 5-H (27)), 3.45 (0.4H, d, J¼11.7 Hz, 5-H
(18b)), 4.25 (0.6H, qn, J¼6.6 Hz, Ala a-H (27)), 4.53
(0.4H, qn, J¼7.0 Hz, Ala a-H (18b)), 4.87 (0.4H, d,
J¼5.1 Hz, 4-H (18b)), 5.36 (0.6H, d, J¼8.6 Hz, NH (27)),
5.59 (0.6H, d, J¼5.1 Hz, 4-H (27)), 5.79 (0.4H, d,
J¼8.2 Hz, NH (18b)); MS m/z 333.1494 (M+H)
(C14H25N2O5S requires 333.1484), 277 (MꢁMe2C]CH2).
8.29. 6S,8aR-3,3,6-Trimethyltetrahydrothiazolo[3,4-a]-
pyrazine-5,8-dione (23b)
Compound 22b (190 mg, 480 mmol) was stirred with Et3N
(97 mg, 1.0 mmol) in CH2Cl2 (5.0 mL) for 5 min. Evapora-
tion and chromatography (EtOAc) afforded 23b (15 mg,
15%) as a white solid: mp 155.8 ꢀC (DSC); IR nmax 3436,
1693, 1652 cmꢁ1; NMR dH 1.46 (3H, d, J¼7.0 Hz, 6-Me),
1.85 (3H, s, 3-Me), 1.89 (3H, s, 3-Me), 3.25 (1H, dd,
J¼12.5, 6.6 Hz, 1a-H), 3.30 (1H, dd, J¼12.5, 10.2 Hz,
1b-H), 4.07 (1H, ddq, J¼7.0, 1.0, 0.8 Hz, 6-H), 4.57 (1H,
dddd, J¼10.5, 6.6, 1.6, 0.8 Hz, 8a-H), 7.28 (1H, br, NH);
MS m/z 368 (M+mNBA), 215.0862 (M+H) (C9H15N2O2S
requires 215.0854). Found C, 50.43; H, 6.55; N, 12.9.
C9H14N2O2S requires C, 50.45; H, 6.58; N, 13.07%.
8.33. Pentafluorophenyl R-2,2-dimethyl-3-(N-(1,1-di-
methylethoxycarbonyl)-D-alanyl)tetrahydrothiazole-4-
carboxylate (28) and pentafluorophenyl R-2,2-dimethyl-
3-(N-(1,1-dimethylethoxycarbonyl)-L-alanyl)tetra-
hydrothiazole-4-carboxylate (19b)
8.30. 6S,8aR-3,3-Dimethyl-6-(1-methylethyl)tetra-
hydrothiazolo[3,4-a]pyrazine-5,8-dione (23c)
The above mixture of 27 and 18b was treated with penta-
fluorophenol and DCC, as for the synthesis of 19a, to give
a mixture of 28 and 19b (80%) as a colourless gum: NMR
dH 1.30 (2.1H, d, J¼6.6 Hz, Ala-Me (28)), 1.37 (0.9H, d,
J¼6.6 Hz, Ala-Me (19b)), 1.39 (2.7H, s, But (19b)), 1.43
(6.3H, s, But (28)), 1.85 (0.9H, s, 2-Me (19b)), 1.87 (2.1H,
s, 2-Me (28)), 1.89 (2.1H, s, 2-Me (28)), 1.94 (0.9H, s, 2-
Me (19b)), 3.37 (0.3H, dd, J¼12.5, 3.5 Hz, 5-H (28)), 3.40
(0.7H, dd, J¼12.5, 5.9 Hz, 5-H (28)), 3.49 (1H, d,
J¼12.5 Hz, 5-H), 4.22 (0.7H, qn, J¼7.0 Hz, Ala a-H
(28)), 4.38 (0.3H, m, Ala a-H (19b)), 5.01 (0.7H, d,
J¼9.0 Hz, NH (28)), 5.19 (0.7H, d, J¼5.5 Hz, 4-H (28)),
5.58 (0.3H, d, J¼3.5 Hz, 4-H (19b)), 6.13 (0.3H, t,
J¼3.9 Hz, NH (19b)); NMR dF ꢁ163.8 (2F, t, J¼21.0 Hz,
30,50-F2), ꢁ162.1 (2F, t, J¼21.0 Hz, 30,50-F2), ꢁ161.5 (2F,
m, 30,50-F2), ꢁ161.1 (2F, t, J¼22.4 Hz, 30,50-F2), 156.4
(1F, t, J¼21.0 Hz, 40-F), ꢁ156.6 (1F, t, J¼21.0 Hz, 40-F),
157.1 (1F, t, J¼21.0 Hz, 40-F), ꢁ157.8 (1F, t, J¼21.0 Hz,
40-F), ꢁ150.9 (2F, d, J¼17.1 Hz, 20,60-F2), ꢁ151.9 (2F, d,
J¼17.1 Hz, 20,60-F2), ꢁ152.1 (2F, d, J¼18.4 Hz, 20,60-F2),
ꢁ152.6 (2F, d, J¼17.1 Hz, 20,60-F2); MS m/z 499.1340
(M+H) (C20H24F5N2O5S requires 499.1326), 443
(MꢁMe2C]CH2), 425 (MꢁButO).
Compound 22c was treated with Et3N, as for the synthesis of
23b except that the reaction time was 10 min, to give 23c
(87%) as a white solid: mp 186.4 ꢀC (DSC); IR nmax 3213,
1692 cmꢁ1; NMR ((CD3)2SO) dH 0.83 (3H, d, J¼7.0 Hz,
CHCH3), 1.0 (3H, d, J¼7.0 Hz, CHCH3), 1.76 (3H, s,
3-Me), 1.77 (3H, s, 3-Me), 2.34 (1H, d septet, J¼7.0,
2.3 Hz, CHMe2), 3.14 (1H, dd, J¼11.7, 10.2 Hz, 1b-H),
3.23 (1H, dd, J¼11.7, 5.9 Hz, 1a-H), 3.34 (1H, br, NH),
3.89 (1H, dd, J¼2.3, 1.2 Hz, 6-H), 4.64 (1H, ddd, J¼11.7,
5.9, 1.2 Hz, 8a-H); MS m/z 396 (M+mNBA), 243.1168
(M+H) (C11H19N2O2S requires 243.1167).
8.31. 6S,8aR-3,3-Dimethyl-6-phenylmethyltetrahydro-
thiazolo[3,4-a]pyrazine-5,8-dione (23d)
Compound 22d was treated with Et3N, as for the synthesis of
23b except that the reaction time was 1.5 h, to give 23d
(81%) as a colourless gum: IR (film) nmax 3377, 1794,
1688 cmꢁ1; NMR dH 1.87 (3H, s, 3-Me), 1.92 (3H, s,
3-Me), 2.82 (1H, dd, J¼14.4, 10.2 Hz, PhCH), 3.19 (1H,
dd, J¼12.1, 10.5 Hz, 1b-H), 3.26 (1H, dd, J¼12.1, 5.9 Hz,
1a-H), 3.56 (1H, dd, J¼14.4, 3.9 Hz, PhCH), 4.24 (1H,
ddd, J¼10.2, 3.9, 0.8 Hz, 6-H), 4.53 (1H, dddd, J¼11.3,
5.9, 1.6, 0.8 Hz, 8a-H), 5.82 (1H, s, NH), 7.21–7.37 (5H,
m, Ph-H5); MS m/z 291.1169 (M+H) (C15H18N2O2S
requires 291.1167).
8.34. R-(2,2-Dimethyl-3-(N-(1,1-dimethylethoxycar-
bonyl)-D-alanyl)-N-(2-(4-nitrophenyl)ethyl)tetrahydro-
thiazole-4-carboxamide (29) and R-2,2-dimethyl-3-(N-
(1,1-dimethylethoxycarbonyl)-L-alanyl)-N-(2-(4-nitro-
phenyl)ethyl)tetrahydrothiazole-4-carboxamide (20b)
8.32. R-2,2-Dimethyl-3-(N-(1,1-dimethylethoxycar-
bonyl)-D-alanyl)tetrahydrothiazole-4-carboxylic acid
(27) and R-2,2-dimethyl-3-(N-(1,1-dimethylethoxycar-
bonyl)-L-alanyl)tetrahydrothiazole-4-carboxylic acid
(18b)
The above mixture of 28 and 19b was treated with 24$HCl
and Et3N, as for the synthesis of 20b, to give a mixture of
29 and 20b (220 mg, 76%) as a white solid: mp 69–80 ꢀC;
NMR ((CD3)2SO) dH 0.93 (1.8H, d, J¼6.6 Hz, Ala-Me
(29)), 1.11 (1.2H, d, J¼6.6 Hz, Ala-Me (20b)), 1.36 (3.6H,
s, But (20b)), 1.37 (5.4H, s, But (20b)), 1.59 (1.2H, s, 2-
Me (20b)), 1.68 (1.8H, s, 2-Me (29)), 1.70 (1.8H, s, 2-Me
A mixture of Boc(D-Ala)F 26 (308 mg, 1.6 mmol) and
17b (154 mg, 800 mmol) was stirred with 11 (500 mg,

11262
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
(29)), 1.71 (1.2H, s, 2-Me (20b)), 2.88 (0.8H, m, ArCH2
(20b)), 2.95 (1.2H, m, ArCH2 (29)), 3.00–4.00 (5H, m,
5-H2, NHCH2, Ala a-H), 4.72 (0.4H, d, J¼4.1 Hz, 4-H
(20b)), 5.25 (0.6H, d, J¼4.3 Hz, 4-H (29)), 7.05 (0.4H, d,
J¼5.9 Hz, NH (20b)), 7.13 (0.6H, d, J¼7.4 Hz, NH (29)),
7.49 (0.8H, d, J¼8.2 Hz, Ar 2,6-H2 (20b)), 7.51 (1.2H, d,
J¼8.6 Hz, Ar 2,6-H2 (29)), 8.13 (2H, d, J¼8.6 Hz, Ar
3,5-H2), 8.17 (1H, d, J¼5.9 Hz, Ala NH); MS m/z
481.2128 (M+H) (C22H33N4O6S requires 481.2121), 425
(MꢁMe2C]CH2), 381 (MꢁBoc).
4.85 (1H, d, J¼9.4 H, ValNH), 5.07 (0.2, d, J¼6.2 Hz,
4-H), 5.36 (0.8H, d, J¼6.6 Hz, 4-H), 6.03 (0.2H, br,
NHCH2), 6.41 (0.8H, br, NHCH2), 7.35 (0.4H, d, J¼
8.6 Hz, Ar 2,6-H2), 7.40 (1.6H, d, J¼8.6 Hz, Ar 2,6-H2),
8.15 (0.4H, d, J¼9.0 Hz, Ar 3,5-H2), 8.17 (1.6H, d,
J¼8.6 Hz, Ar 3,5-H2); MS m/z 509.2437 (M+H)
(C24H37N4O6S requires 509.2434).
8.38. S-2,2-Dimethyltetrahydrothiazole-4-carboxylic
acid hydrochloride (36)
8.35. S-2,2-Dimethyl-3-(N-(1,1-dimethylethoxycar-
bonyl)-D-valinyl)tetrahydrothiazole-4-carboxylic
acid (32)
D-Cys$HCl$H2O 35 was treated with acetone and 2,2-di-
methoxypropane, as for the synthesis of 11, to give 36
(78%) as a white solid: mp 166–170 ꢀC (lit.39 mp 165–
168 ꢀC for R enantiomer); IR nmax 3412, 2600, 1743 cmꢁ1
;
Compound 31 (prepared as 17c) (270 mg, 1.1 mmol) was
stirred with 11 (250 mg, 1.1 mmol) and Pri2NEt (310 mg,
2.4 mmol) in dry DMF (10 mL) for 16 h. The evaporated
residue, in EtOAc, was washed with cold citric acid, cold
H2O and brine. Drying, evaporation and chromatography
(hexane/EtOAc/AcOH, 49:49:2) afforded 32 (200 mg,
50%) as a colourless gum: IR (film) nmax 3329, 1714, 1657,
1462 cmꢁ1; NMR dH 0.92 (3H, d, J¼6.2 Hz, Val-Me), 0.94
(3H, d, J¼6.6 Hz, Val-Me), 1.43 (9H, s, But), 1.85 (3H, s,
2-Me), 1.89 (3H, s, 2-Me), 2.05 (1H, m, Val b-H), 3.27
(1H, dd, J¼12.1, 5.7 Hz, 5-H), 3.40 (1H, d, J¼12.1 Hz,
5-H), 3.86 (1H, t, J¼9.6 Hz, Val a-H), 5.42 (1H, d,
J¼10.1 Hz, NH), 5.66 (1H, d, J¼5.7 Hz, 4-H); MS m/z
361.1810 (M+H) (C16H29N2O5S requires 361.1797), 305
(MꢁMe2C]CH2).
NMR (D2O) dH 2.06 (6H, s, 2ꢂMe), 2.92 (1H, dd, J¼15.2,
3.9 Hz, 5-H), 3.00 (1H, dd, J¼15.2, 5.9 Hz, 5-H), 4.06
(1H, dd, J¼5.9, 3.9 Hz, 4-H).
8.39. S-2,2-Dimethyl-3-(N-(1,1-dimethylethoxycar-
bonyl)-L-valinyl)tetrahydrothiazole-4-carboxylic acid
(37c)
Compound 17c (1.08 g, 4.6 mmol) was stirred with 36
(1.00 g, 5.1 mmol) and Pr2i NEt (1.24 g, 9.6 mmol) in dry
DMF (100 mL) for 16 h. The evaporated residue, in EtOAc,
was washed with cold citric acid, cold H2O and brine. Dry-
ing, evaporation and chromatography (hexane/EtOAc/
AcOH, 70:28:2) afforded 37c (630 mg, 35%) as a colourless
gummy solid: IR nmax 3434, 2970, 1723, 1605, 1513 cmꢁ1
;
NMR dH 0.87–0.95 (6H, m, 2ꢂVal-Me), 1.43 (9H, s, But),
1.85 (3H, s, 2-Me), 1.88 (3H, s, 2-Me), 2.00 (1H, m, Val
b-H), 3.20–3.50 (2H, m, 5-H+Val a-H), 3.85 (1H, t,
J¼9.8 Hz, 5-H), 5.36 (1H, d, J¼10.2 Hz, 4-H), 5.68 (0.7H,
d, J¼5.1 Hz, NH), 8.63 (1H, br, OH); MS m/z 361.1810
(M+H) (C16H29N2O5S requires 361.1797), 305 (Mꢁ
Me2C]CH2).
8.36. Pentafluorophenyl R-2,2-dimethyl-3-(N-(1,1-
dimethylethoxycarbonyl)-D-valinyl)tetrahydrothiazole-
4-carboxylate (33)
Compound 32 was treated with pentafluorophenol and DCC,
as for the synthesis of 19a, to give 33 (41%) as a colourless
oil: NMR dH 0.89 (2.1H, d, J¼7.0 Hz, Val-Me), 0.98 (2.1H,
d, J¼6.6 Hz, Val-Me), 1.04 (0.9H, d, J¼7.0 Hz, Val-Me),
1.10 (0.9H, d, J¼7.0 Hz, Val-Me), 1.45 (6.3H, s, But), 1.48
(2.7H, s, But), 1.89 (2.1H, s, 2-Me), 1.93 (2.1H, s, 2-Me),
2.00 (0.9H, s, 2-Me), 2.02 (1H, m, Val b-H), 2.06 (0.9H, s,
2-Me), 3.30 (0.3H, dd, J¼12.9, 3.1 Hz, 5-H), 3.43 (0.3H,
d, J¼4.7 Hz, 5-H), 3.54 (0.7H, dd, J¼12.5, 6.2 Hz, 5-H),
3.83 (0.7H, t, J¼9.4, 5-H), 5.03 (0.7H, d, J¼9.8 Hz, 4-H),
5.08 (0.3H, d, J¼9.0 Hz, 4-H), 5.62 (0.3H, dd, J¼6.6,
2.3 Hz, Val a-H), 5.67 (0.7H, dd, J¼3.9, 2.0 Hz, Val a-H),
6.2 (1H, m, NH).
8.40. S-2,2-Dimethyl-3-(N-(1,1-dimethylethoxycar-
bonyl)-L-leucyl)tetrahydrothiazole-4-carboxylic acid
(37e)
Compound 45 (1.5 g, 3.8 mmol) was stirred with 36
(833 mg, 4.2 mmol) and Pr2i NEt (1.63 g, 12.6 mmol) in dry
DMF (40 mL) for 16 h. The evaporated residue, in EtOAc,
was washed with cold 5% aq citric acid, cold H2O and brine.
Drying and evaporation afforded crude 37e (2.4 g) as
a gummy oil: NMR dH 0.81–0.90 (6H, m, 2ꢂLeu-Me),
1.40 (9H, s, But), 1.50–1.60 (2H, m, Leu b-H2+Leu g-H),
1.83 (3H, s, 2-Me), 1.86 (3H, s, 2-Me), 3.28 (2H, m,
5-H2), 4.17 (1H, m, Leu a-H), 5.30 (1H, d, J¼5.92 Hz,
NH), 5.70 (1H, m, 4-H); MS m/z 375.1948 (M+H)
(C17H31N2O5S requires 375.1954), 319 (MꢁMe2C]CH2).
8.37. R-(2,2-Dimethyl-3-(1,1-dimethylethoxycarbonyl)-
D-valinyl)-N-(2-(4-nitrophenyl)ethyl)tetrahydro-
thiazole-4-carboxamide (34)
Compound 33 was treated with 24$HCl and Et3N, as for the
synthesis of 20c, to give 34 (17%) as a colourless gum: IR
(film) nmax 3320, 1702, 1655, 1514 cmꢁ1; NMR dH 0.83
(3H, d, J¼6.6 Hz, Val-Me), 0.92 (3H, d, J¼6.6 Hz, Val-
Me), 1.42 (7.2H, s, But), 1.55 (1.8H, s, But), 1.79 (2.4H, s,
2-Me), 1.81 (2.4H, s, 2-Me), 1.88 (0.6H, s, 2-Me), 1.91
(0.6H, s, 2-Me), 1.92 (1H, m, Val b-H), 2.97 (2H, t,
J¼7.0 Hz, ArCH2), 3.04 (1H, d, J¼12.5 Hz, 5a-H), 3.36
(1H, dd, J¼12.5, 7.0 Hz, 5b-H), 3.44 (1H, m, ArCH2CH),
3.80 (1H, m, ArCH2CH), 3.86 (1H, t, J¼9.0 Hz, Val a-H),
8.41. Pentafluorophenyl S-2,2-dimethyl-3-(N-(1,1-di-
methylethoxycarbonyl)-L-valinyl)tetrahydrothiazole-
4-carboxylate (38c)
Compound 37c was treated with pentafluorophenol and
DCC, as for the synthesis of 19a, to give gave 38c (68%)
as a colourless oil: NMR dH 0.88 (3H, d, J¼6.6 Hz, Val-
Me), 0.97 (3H, d, J¼6.6 Hz, Val-Me), 1.45 (9H, s, But),
1.88 (3H, s, 2-Me), 1.92 (3H, s, 2-Me), 2.04 (1H, m, Val

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11263
b-H), 3.55 (1H, dd, J¼12.5, 6.6 Hz, 5-H), 3.82 (1H, t,
J¼9.4 Hz, 5-H), 5.08 (1H, br d, J¼9.8 Hz, 4-H), 5.61 and
5.66 (1H, 2ꢂd, J¼4.7 Hz, Val a-H), 6.22 (1H, br d,
J¼3.9 Hz, NH); MS m/z 527.1656 (M+H) (C22H28F5N2O5S
requires 527.1639), 471 (MꢁButO).
J¼8.6 Hz, LeuNH), 5.30 (0.2H, d, J¼7.0 Hz, 4-H), 5.42
(0.8H, d, J¼7.0 Hz, 4-H), 6.15 (1H, t, J¼5.1 Hz, NHCH2),
7.41 (2H, d, J¼8.6 Hz, Ar 2,6-H2), 8.15 (0.4H, d,
J¼8.3 Hz, Ar 3,5-H2), 8.19 (1.6H, d, J¼8.6 Hz, Ar 3,5-
H2); MS m/z 523.2596 (M+H) (C25H39N4O6S requires
523.2590), 467 (MꢁMe2C]CH2), 423 (MꢁBoc).
8.42. Pentafluorophenyl S-2,2-dimethyl-3-(N-(1,1-di-
methylethoxycarbonyl)-L-leucyl)tetrahydrothiazole-4-
carboxylate (38e)
8.45. S-2,2-Dimethyl-N-(2-(4-nitrophenyl)ethyl)-3-
(L-valinyl)tetrahydrothiazole-4-carboxamide tri-
fluoroacetate salt (40c)
Compound 37e was treated with pentafluorophenol and
DCC, as for the synthesis of 19a, to give crude 38e as
a pale yellow oil: NMR dH 0.88 (3H, d, J¼6.4 Hz, Leu-Me),
0.90 (3H, d, J¼6.4 Hz, Leu-Me), 1.44 (9H, s, But), 1.55–
1.65 (2H, m, Leu b,g-H3), 1.86 (3H, s, 2-Me), 1.91 (3H, s,
2-Me), 3.47 (1H, dd, J¼12.7, 9.8 Hz, 5b-H), 3.54 (1H, dd,
J¼12.7, 4.8 Hz, 5a-H), 4.17 (1H, dt, J¼2.7, 8.7 Hz, Leu
a-H), 4.96 (1H, d, J¼9.0 Hz, 4-H), 6.17 (1H, d, J¼9.0 Hz,
NH); NMR dF ꢁ161.5 (2F, t, J¼17.1 Hz, 30,50-F2), ꢁ156.6
(1F, t, J¼22.4 Hz, 40-F), ꢁ152.1 (2F, d, J¼18.4 Hz,
20,60-F2). MS m/z 541.1797 (M+H) (C23H30N2O5F5S
requires 541.1796), 485 (MꢁMe2C]CH2), 441 (MꢁBoc).
Compound 39c was treated with CF3CO2H, as for the syn-
thesis of 21c, to give 40c (quant.) as a highly hygroscopic
viscous oil: NMR ((CD3)2SO) dH 0.82 (3H, d, J¼7.4 Hz,
Val-Me), 0.84 (3H, d, J¼7.0 Hz, Val-Me), 1.80 (6H, s,
2ꢂ2-Me), 2.02 (1H, m, Val b-H), 2.88 (2H, t, J¼7.0 Hz,
ArCH2), 3.08 (1H, d, J¼12.1 Hz, 5-H), 3.38 (2H, m,
NHCH2), 3.52 (1H, dd, J¼12.1, 4.7 Hz, 5-H), 4.60 (1H, m,
Val a-H), 5.05 (1H, d, J¼5.9 Hz, 4-H), 7.49 (2H, d,
J¼8.6 Hz, Ar 2,6-H2), 8.15 (2H, d, J¼8.6 Hz, Ar 3,5-H2),
8.23 (1H, t, J¼5.9 Hz, NH); MS m/z 409.1911 (M+H)
(C19H29N4O4S requires 409.1910).
8.43. S-(2,2-Dimethyl-3-(1,1-dimethylethoxycarbonyl)-
L-valinyl)-N-(2-(4-nitrophenyl)ethyl)tetrahydro-
thiazole-4-carboxamide (39c)
8.46. S-2,2-Dimethyl-N-(2-(4-nitrophenyl)ethyl)-3-
(L-leucyl)tetrahydrothiazole-4-carboxamide tri-
fluoroacetate salt (40e)
Compound 38c was treated with 24$HCl and Et3N, as for the
synthesis of 20c, to give 39c (49%) as a colourless oil: IR
nmax 3323, 1698, 1605, 1520 cmꢁ1; NMR dH 0.83 (3H, d,
J¼6.6 Hz, Val-Me), 0.92 (3H, d, J¼6.6 Hz, Val-Me), 1.42
(7.2H, s, But), 1.55 (1.8H, s, But), 1.80 (2.4H, s), 1.82
(2.4H, s), 1.89 (0.6H, s) and 1.91 (0.6H, s, 2-Me2), 1.95
(1H, m, Val b-H), 2.81 (1.6H, dt, J¼14.1, 7.0 Hz, ArCH2),
3.04 (0.8H, d, J¼12.5 Hz, 5b-H), 3.36 (1H, dd, J¼12.5,
6.6 Hz, 5a-H), 3.44 (1H, m, ArCH2CH), 3.77 (1H, m,
ArCH2CH), 3.86 (1H, t, J¼9.0 Hz, Val a-H), 4.92 (1H, d,
J¼9.8 Hz, Val NH), 5.07 (0.2H, d, J¼7.0 Hz, 4-H),
5.36 (0.8H, d, J¼6.2 Hz, 4-H), 6.09 (0.2H, br, NHCH2),
6.47 (0.8H, t, J¼5.5 Hz, NHCH2), 7.35 (0.4H, d,
J¼8.6 Hz, Ar 2,6-H2), 7.40 (1.6H, d, J¼8.6 Hz, Ar
2,6-H2), 8.13 (0.4H, d, J¼8.2 Hz, Ar 3,5-H2), 8.16 (1.6H,
d, J¼8.6 Hz, Ar 3,5-H2); MS m/z 509.2432 (M+H)
(C24H37N4O6S requires 509.2434), 453 (MꢁButO). Some
1H NMR peaks from the minor isomer overlapped with
other peaks from the major isomer and thus were not iden-
tifiable.
Compound 39e was treated with CF3CO2H, as for the
synthesis of 21c, to give 40e (quant.) as colourless highly
hygroscopic gum: NMR (CD3OD) dH 0.90 (3H, d,
J¼4.7 Hz, Leu-Me), 0.98 (3H, d, J¼4.3 Hz, Leu-Me),
1.60–1.70 (3H, m, Leu b,g-H3), 1.89 (3H, s, 2-Me), 1.92
(3H, s, 2-Me), 2.95–3.07 (3H, m, 5-H, ArCH2), 3.38–3.52
(2H, m, ArCH2CH, 5-H), 3.70 (1H, m, ArCH2CH), 3.95
(1H, m, Leu a-H), 5.08 (1H, d, J¼6.2 Hz, 4-H), 7.52 (2H,
d, J¼7.0 Hz, Ar 2,6-H2), 8.20 (2H, d, J¼7.4 Hz, Ar 3,5-
H2); MS m/z 423.2077 (M+H) (C20H31N4O4S requires
423.2066).
8.47. 6S,8aS-3,3-Dimethyl-6-(1-methylethyl)tetrahydro-
thiazolo[3,4-a]pyrazine-5,8-dione (41c)
Compound 38c was treated with HCl, as for the synthesis of
22b, to give crude pentafluorophenyl S-2,2-dimethyl-3-
(N-(L-valinyl)tetrahydrothiazole-2-carboxylate hydrochlo-
ride (quant.) as a pale yellow gummy solid: IR nmax
3412 cmꢁ1; NMR ((CD3)2SO) dH 0.88 (3H, d, J¼6.6 Hz,
Val-Me), 0.92 (3H, d, J¼7.0 Hz, Val-Me), 1.78 (3H, s,
2-Me), 1.82 (3H, s, 2-Me), 2.06 (1H, m, Val b-H), 3.11
(1H, dd, J¼11.7, 10.9 Hz, 5-H), 3.22 (1H, dd, J¼11.7,
5.5 Hz, 5-H), 3.32 (3H, br, N+H3), 4.64 (1H, dd, J¼10.9,
5.5 Hz, 4-H), 5.19 (1H, m, Val a-H); NMR dF ꢁ171.46 (2F,
m, 30,50-F2), ꢁ165.12 (1F, dt, J¼23.7 Hz, 40-F), ꢁ161.53
(2F, dd, J¼19.7, 6.6 Hz, 20,60-F2); MS m/z 427 (M+H), 243
(MꢁC6F5O). This material was treated with Et3N, as for
the synthesis of 23b, to give 41c (61%) as a white solid:
mp 180–182 ꢀC; NMR ((CD3)2SO) dH 0.88 (3H, d,
J¼6.6 Hz, 6-CMe), 0.91 (3H, d, J¼6.6 Hz, 6-CMe), 1.78
(3H, s, 3-Me), 1.82 (3H, s, 3-Me), 2.07 (1H, m, 6-CH),
3.11 (1H, dd, J¼11.7, 10.5 Hz, 1a-H), 3.23 (1H, dd, J¼11.7,
5.5 Hz, 1b-H), 3.40 (1H, dd, J¼5.9, 3.5 Hz, 6-H), 4.64 (1H,
dd, J¼10.9, 5.5 Hz, 8a-H), 8.90 (1H, d, J¼3.5 Hz, NH); MS
m/z 396 (M+mNBA), 243.1176 (M+H) (C11H19N2O2S
8.44. S-(2,2-Dimethyl-3-(1,1-dimethylethoxycarbonyl)-
L-leucyl)-N-(2-(4-nitrophenyl)ethyl)tetrahydrothiazole-
4-carboxamide (39e)
Compound 38e was treated with 24$HCl and Et3N, as for the
synthesis of 20c, to give 39e (58%) as a white solid: mp 108–
109 ꢀC; NMR dH 0.84 (2.4H, d, J¼6.4 Hz, Leu-Me), 0.90
(2.4H, d, J¼6.4 Hz, Leu-Me), 0.98 (1.2H, d, J¼6.4 Hz,
2ꢂLeu-Me), 1.41 (7.2H, s, But), 1.43 (1.8H, s, But), 1.60–
1.70 (3H, m, Leu b,g-H3), 1.78 (2.4H, s, 2-Me), 1.80
(2.4H, s, 2-Me), 1.84 (0.6H, s, 2-Me), 1.85 (0.6H, s, 2-Me),
2.97 (2H, t, J¼7.0 Hz, ArCH2), 3.05 (1H, d, J¼12.3 Hz,
5b-H), 3.45 (2H, m, ArCH2CH, 5a-H), 3.85 (1H, m,
ArCH2CH), 4.11 (0.8H, m, Leu a-H), 4.30 (0.2H, m, Leu
a-H), 4.81 (0.8H, d, J¼8.6 Hz, LeuNH), 4.92 (0.2H, d,

11264
G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
requires 243.1167). Found C, 54.60; H, 7.39; N, 11.4.
C11H18N2O2S requires C, 54.52; H, 7.49; N, 11.56%.
8.50. S-2,2-Dimethyl-3-(N-(fluoren-9-ylmethoxycar-
bonyl)-L-valinyl)tetrahydrothiazole-4-carboxylic acid
(44)
8.48. 6S,8aS-3,3-dimethyl-6-(2-methylpropyl)tetra-
hydrothiazolo[3,4-a]pyrazine-5,8-dione (41e)
Compound 43 (1.16 g, 2.3 mmol), in dry THF (10 mL), was
added slowly to 36 (495 mg, 2.5 mmol) and Pri2NEt
(970 mg, 7.5 mmol) in dry DMF (30 mL) at 0 ꢀC under N2
for 3 h. The mixture was then slowly warmed to 20 ꢀC and
stirred for 16 h. The evaporated residue, in EtOAc, was
washed with cold 5% aq citric acid and brine. Drying,
evaporation and chromatography (hexane/EtOAc/AcOH,
25:25:1) afforded 44 (600 mg, 50%) as a white solid: mp
95–97 ꢀC; NMR dH 0.92 (3H, d, J¼7.0 Hz, Val-Me), 0.94
(3H, d, J¼6.6 Hz, Val-Me), 1.84 (3H, s, 2-Me), 1.89 (3H,
s, 2-Me), 2.10 (1H, m, Val b-H), 3.25 (1H, dd, J¼12.1,
5.5 Hz, 5-H), 3.35 (1H, d, J¼12.5 Hz, 5-H), 3.90 (1H, t,
J¼9.8 Hz, Val a-H), 4.20 (1H, t, J¼7.0 Hz, CHCH2O),
4.37 (1H, dd, J¼10.9, 7.0 Hz, CHO), 4.45 (1H, dd,
J¼10.5, 7.0 Hz, CHO), 5.43 (1H, d, J¼10.1 Hz, NH), 5.52
(1H, d, J¼5.1 Hz, 4-H), 7.28 (2H, t, J¼7.4 Hz, Ar-H2),
7.38 (2H, t, J¼7.4 Hz, Ar-H2), 7.54 (2H, d, J¼6.6 Hz,
Ar-H2), 7.75 (2H, d, J¼7.4 Hz, Ar-H2).
Compound 38e (700 mg, 1.30 mmol) was stirred with
CF3CO2H (1 mL) and CH2Cl2 (4 mL) for 20 min. Evapora-
tion afforded crude pentafluorophenyl S-2,2-dimethyl-3-
(N-(L-leucyl)tetrahydrothiazole-2-carboxylate trifluoroacetate
salt (quant.) as a gummy solid. This material (554 mg,
1 mmol) was stirred with Et3N (202 mg, 2 mmol) in
CH2Cl2 (3.0 mL) for 30 min. Evaporation and chromato-
graphy (EtOAc) afforded 41e (150 mg, 59%) as a white
solid: mp 153–155 ꢀC; NMR dH 0.95 (3H, d, J¼6.6 Hz,
CHCH3), 1.00 (3H, d, J¼6.6 Hz, CHCH3), 1.60–1.78 (3H,
m, 6-CH2CH), 1.88 (3H, s, 3-Me), 1.89 (3H, s, 3-Me),
3.23 (1H, dd, J¼12.1, 10.9 Hz, 1a-H), 3.32 (1H, dd,
J¼12.1, 5.9 Hz, 1b-H), 3.87 (1H, dt, J¼10.0, 5.0 Hz, 6-H),
4.55 (1H, dd, J¼10.5, 5.5 Hz, 8a-H), 6.29 (1H, br, NH);
MS m/z 257.1316 (C12H21N2O2S requires 257.1324).
8.49. N-(Fluoren-9-ylmethoxycarbonyl)-L-valine penta-
fluorophenyl ester (43)
8.51. N-(1,1-Dimethylethoxycarbonyl)-L-leucine penta-
fluorophenyl ester (45)
FmocValOH 42 was treated with pentafluorophenol and
DCC, as for the synthesis of 19a, to give 43 (70%) as a white
solid: mp 114–117 ꢀC (lit.40 mp 122–123 ꢀC); NMR dH 1.03
(3H, d, J¼6.6 Hz, Val-Me), 1.10 (3H, d, J¼6.6 Hz, Val-Me),
2.40 (1H, m, Val b-H), 4.24 (1H, t, J¼6.6 Hz, CHCH2O),
4.46 (2H, d, J¼6.6 Hz, CH2O), 4.67 (1H, dd, J¼9.4,
5.1 Hz, Val a-H), 5.27 (1H, d, J¼9.4 Hz, NH), 7.30 (2H, t,
J¼7.4 Hz, Ar-H2), 7.38 (2H, t, J¼7.4 Hz, Ar-H2), 7.58
(2H, d, J¼7.4 Hz, Ar-H2), 7.75 (2H, d, J¼7.8 Hz, Ar-H2);
NMR dF ꢁ161.2 (2F, t, J¼20.9 Hz, 30,50-F2), ꢁ156.6 (1F,
t, J¼20.9 Hz, 40-F), ꢁ151.5 (2F, d, J¼18.0 Hz, 20,60-F2).
Boc-L-LeuOH 16e was treated with pentafluorophenol and
DCC, as for the synthesis of 19a, to give 45 (79%) as a col-
ourless oil (lit.41 oil): NMR dH 1.02 (6H, d, J¼6.3 Hz,
2ꢂLeu-Me), 1.47 (9H, s, But), 1.67 (2H, dd, J¼9.7,
8.2 Hz, Leu b-H), 1.8 (2H, m, Leu b-H+Leu g-H), 4.41
(0.1H, m, Leu a-H), 4.62 (0.9H, m, Leu a-H), 5.75 (0.1H,
br, NH), 4.92 (0.9H, d, J¼8.2 Hz, NH); NMR dF ꢁ162.1
(1.8F, t, J¼21 Hz, 30,50-F2), ꢁ161.9 (0.2F, t, J¼21.0 Hz,
30,50-F2), ꢁ157.6 (0.9F, t, J¼22.4 Hz, 40-F), ꢁ157.4 (0.1F,
t, J¼22.4 Hz, 40-F), ꢁ152.7 (0.2F, d, J¼19.7 Hz, 20,60-F2),
Table 3. Crystal data and structure refinement of 20b, 23b and 23c
Compound
20b
23b
23c
Empirical formula
Formula weight
Crystal system
Space group
C22H32N4O6S
480.58
Monoclinic
P21
7.2780(1)
21.0700(2)
16.5780(2)
—
94.700(1)
—
2533.65(5)
4
1.260
C9H14N2O2S
214.28
Orthorhombic
P212121
6.1600(1)
6.5280(1)
25.9050(5)
—
C11H18N2O2S
242.33
Triclinic
P1
˚
a/A
5.7440(2)
9.7410(3)
11.0190(4)
91.399(1)
94.980(1)
98.341(2)
607.29(4)
2
˚
b/A
˚
c/A
a/ꢀ
b/ꢀ
g/ꢀ
—
—
3
˚
U/A
Z
1041.70(3)
4
1.366
0.287
456
0.40ꢂ0.20ꢂ0.10
4.06, 27.86
ꢁ8ꢃhꢃ8; ꢁ8ꢃkꢃ8; ꢁ34ꢃlꢃ34
15756
2475, 0.0470
2180
2475/1/136
1.037
0.0298, 0.0697
0.0386, 0.0730
0.02(8)
Dc/g cmꢁ3
1.325
0.255
260
M/mmꢁ1
F(000)
Crystal size/mm
0.170
1024
0.22ꢂ0.08ꢂ0.08
3.81, 27.47
ꢁ9ꢃhꢃ9; ꢁ27ꢃkꢃ27; ꢁ21ꢃlꢃ21
48362
11591, 0.0738
7928
11591/5/625
1.005
0.0451, 0.0789
0.0886, 0.0904
0.01(5)
0.15ꢂ0.10ꢂ0.10
3.60, 27.45
ꢁ7ꢃhꢃ7; ꢁ12ꢃkꢃ12; ꢁ14ꢃlꢃ14
9382
5031, 0.0391
4185
5031/5/307
1.040
0.0364, 0.0760
0.0513, 0.0818
0.04(5)
Theta min, max/ꢀ
Index ranges
Reflections collected
Independent reflections, R(int)
Reflections observed (>2)
Data/restraints/parameters
Goodness-of-fit on F2
Final R1, wR2 indices [I>2(I)]
Final R1, wR2 indices (all data)
Flack parameter
ꢁ3
˚
Largest diff. peak and hole/eA
0.226, ꢁ0.284
0.192, ꢁ0.219
0.253, ꢁ0.242

G. A. R. Y. Suaifan et al. / Tetrahedron 62 (2006) 11245–11266
11265
ꢁ152.2 (1.8F, d, J¼18.4 Hz, 20,60-F2); MS m/z 795 (2 M),
(Swansea) for some of the mass spectra. We are very grateful
to the Association for International Cancer Research for
generous funding in support of the project.
398.1394 (M+H) (C17H21N1O4S requires 398.1391).
8.52. X-ray crystallography
References and notes
Single crystals of compounds 20b, 23b and 23c were ana-
lysed at 150(2) K using graphite-monochromated Mo Ka
radiation and a Nonius Kappa CCD diffractometer. Details
of the data collections, solutions and refinements are given
in Table 3. The structures were uniformly solved using
SHELXS-9742 and refined using full-matrix least squares
in SHELXL-97.43
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˚
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580), Dionex column thermostat for HPLC and GPC (Model
585) and a Dionex UV–vis detector (Model UVD 170S/
340S). A C18 Hypersil (Thermoquest BDS) column
(250ꢂ4.6 mm) was used. The mobile phase was composed
of a mixture (65/35, v/v) of acetonitrile and aqueous phos-
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with aqueous potassium hydroxide (19 M) using a Hanaa
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with a Schleicher and Schuell filter membrane (pore size
and diameter of 0.45 and 47 mm). The flow rate during the
assays was 0.8 mL minꢁ1 and detection was accomplished
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amide salt 21a, 21b, 21c, 21d, 40c or 40e (1.0 mg) was
added to the buffer (1.0 mL) at the appropriate pH and
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We thank Dr. Steven J. Black (University of Bath) for the
NMR spectra and the EPSRC Mass Spectrometry Centre

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