Efficient microwave assisted syntheses of 2,5-diketopiperazines in aqueous media

Article abstract of DOI:10.3390/molecules14082836

Aqueous in situ one-pot N-Boc-deprotection-cyclization of Nα-Boc-dipeptidyltert-butyl and methyl esters under microwave irradiation afforded 2,5-diketopiperazines (DKPs) in excellent yields. This protocol is rapid, safe, environmentally friendly, and high

Full text of DOI:10.3390/molecules14082836

Molecules 2009, 14, 2836-2849; doi:10.3390/molecules14082836
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Efficient Microwave Assisted Syntheses of 2,5-Diketopiperazines
in Aqueous Media †
Lemuel Pérez-Picaso 1, Jaime Escalante 1, Horacio F. Olivo 2 and María Yolanda Rios 1,*
1
Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Avenida
Universidad 1001, Col. Chamilpa, 62209 Cuernavaca, Morelos, México
2
Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, IA 52242, USA
†
This paper is taken in part from the Ph.D. thesis of Lemuel Pérez-Picaso
* Author to whom correspondence should be addressed; E-mail: myolanda@uaem.mx;
Tel. +52 777 329 70 00 ext. 6024; Fax: +52 777 329 79 97
Received: 23 June 2009; in revised form: 21 July 2009 / Accepted: 23 July 2009 /
Published: 31 July 2009
Abstract: Aqueous in situ one-pot N-Boc-deprotection-cyclization of Nα-Boc-dipeptidyl-
tert-butyl and methyl esters under microwave irradiation afforded 2,5-diketopiperazines
(DKPs) in excellent yields. This protocol is rapid, safe, environmentally friendly, and
highly efficient, and showed that the tert-butoxy moiety is also an excellent leaving group
for these cyclizations.
Keywords: 2,5-diketopiperazines; DKPs; cyclic dipeptides; microwave irradiation
1. Introduction
Cyclic peptides are a very important family of bioactive compounds easily available both from
natural sources (plants, animals or microorganisms) or by means of synthetic methods. Their chemical
properties, like the lack of charges at the amine and carboxylic terminal groups and the lack of
zwitterionic character, confer to these molecules high lipophilicity [1], and fast membrane absorption
in the digestive tract because of their high permeability [2]. The structural rigidity of the cyclic
peptides increases its affinity and selectivity toward protein ligands, making their half-lives in vivo
much greater than those of linear peptides [3]. Among them, 2,5-diketopiperazines (DKPs, also known
as cyclic dipeptides, 2,5-dioxopiperazines, cyclo(dipeptides), or anhydride dipeptides) are the smallest

Molecules 2009, 14
2837
cyclopeptides. These peptides are most commonly found as natural products [4], showing
antimicrobial [5], antitumoral and antiviral [6], cytotoxic [7], and neuroprotective effects [8], among
other activities. Some DKPs are stable to proteolysis (enzymatic degradation), an important feature for
their high activity. All these properties make DKPs an interesting group of molecules for the
development of new therapeutic agents.
The DKP core derives, chemically and biosynthetically, from folding and head-tail cyclization
between N and C-terminal amino acids of linear dipeptides [9]. These heterocyclic compounds possess
two amide groups (with acceptor-donor properties) with the possibility of including up to four
hydrogen bonds [10]. DKPs can be synthesized in solution or in solid phase from commercially
available and appropriately protected chiral α-amino acids. Their syntheses can be carried out with the
appropriate linear dipeptide [11-13], followed by N-deprotection and cyclization using either basic
(aminolysis of dipeptide ester in methanolic ammonia, Fischer method) [10,14], neutral (aminolysis of
dipeptide ester in methanol, autoaminolysis) [15], or acidic conditions (Suzuki method) [16]. Some of
these methods variously result in good reaction yields [17], low reaction yields [18], with or without
epimerization [19].
Nowadays, microwave heating can be used to obtain good results from previously unsuccessful or
low-yielding reactions. Under microwave heating conditions the reaction times can be reduced from
days to hours, from hours to minutes, or from minutes to seconds, and the reaction yields can be
greatly increased [20,21]. DKPs syntheses using microwave assistance has barely been investigated.
Some examples of the use of this methodology are the syntheses of dimeric structures based on
intermolecular DKP formation by activation of C-terminal glycine monomers [22], the solvent-free
synthesis of DKPs in one-pot deprotection-cyclization of N-Boc-dipeptidyl ethyl and methyl esters
[23], and the DKPs formation using dipeptide methyl ester hydrochlorides in water in three [24] and
two steps [25]. In the present study, we report the syntheses of DKPs using Nα-Boc-dipeptidyl methyl
and tert-butyl esters in water under microwave irradiation. DKPs were obtained in excellent yields
without epimerization employing this general and highly efficient protocol.
2. Results and Discussion
The formation of DKPs occurs through an intramolecular aminolysis depending largely on the
nature and the sequence of the amino acids in solution [11]. Under these conditions, when DKP
formation is an undesired reaction, tert-butyl ester protection for the carboxy terminus function is used
to prevent ring closure of dipeptides under basic conditions, because it provides a poor leaving group
and steric hindrance. Nucleophilic removal of this protecting group with ring formation has been
observed only in a few cases [26,27]. We studied the cyclization reaction assisted by microwave
heating with the purpose of investigating the possible use of Nα-Boc-dipeptidyl tert-butyl esters as
DKPs precursors, as part of a synthetic strategy toward the cis-DKP fragment of the natural product
cyclo[N-(Lys-Phe)-Orn-Val] (1, Figure 1) [28]. We report our findings herein.

Molecules 2009, 14
2838
Figure 1. Structure of cyclo[N-(Lys-Phe)-Orn-Val] (1).
NH2
NH2
O
H
N
O
H2N
N
O
NH
O
1
In an effort to explore optimized conditions for the cyclization under microwave irradiation, Boc-
Orn(Cbz)-Val-OtBu (2a) was subjected to different reaction variables, such as solvent, temperature
(T), irradiation power (W), and exposure time exchange (Table 1). Toluene, toluene-isopropanol (1:1)
mixture, and xylene were unsuccessful in the transformation of dipeptide 2a to DKP 3a (entries 1-6).
When DMF was used as solvent at 200 °C, 300 W, and 5 min reaction time, DKP 3a was obtained in
61% yield (entry 7). Water resulted a better solvent for the cyclization (entries 8-11). As can be
observed in Table 1, with water as solvent, 200 °C, 300 W, and 5 min reaction time resulted in the best
conditions, yielding 3a in 89% yield. However, pressure in the sealed reaction tube increased
dramatically due to the generation of CO2 gas, increasing the risk of breaking the reaction vessel.
Increasing the temperature and the reaction time did not improve the reaction yield any further
(entry 11). The optimized conditions were then used in all cyclizations of Nα-Boc-dipeptidyl esters 2a-
2l to DKPs 3a-3k.
Table 1. Cyclization of dipeptide 2a to diketopiperazine 3a under microwave irradiation.a
H
N
O
H
N
O
O
O
O
O
H
N
O
HN
O
N
O
H
NH
O
O
2a
3a
Entry
Solvent
T (°C)
170
170
200
200
200
200
200
200
200
200
250
Power (W) Time (min) 3a (% yield)
1
2
3
4
5
6
7
8
9
160
180
250
300
150
300
300
250
250
300
250
10
10
10
10
10
10
5
1
2.5
5
nr
nr
nr
nr
nr
nr
61
22
85
89
86
toluene
toluene-isopropanol (1:1)
xylene
DMF
H2O
10
11
10
nr = no reaction; a Reactions performed in a monomode microwave CEM Discover apparatus.

Molecules 2009, 14
2839
DKPs 3a-3k were synthesized in two steps: dipeptide formation (starting with Nα-Boc-terminal and
C-OtBu or C-OMe amino acids, to obtain the corresponding Nα-Boc-dipeptidyl esters 2a-2l) and
subsequent cyclization (Table 2).
Table 2. Coupling of amino acids and ring closure under microwave irradiation.a
O
R1
R2
R1
R3
N
R1
R3
O
N
a
b
+
BOCHN
COOH
HN
R3
COOR4
BOCHN
OR4
HN
R2
O
R2
O
2a-l
3a-k
(a) EDAC, HOBt, DMAP, TEA/CH2Cl2, 5 °C, then overnight at rt; (b) H2O (1 mL), MW (250 °C, 250 W and
150 psi) for 10 min.
Entry
R1
(S)-Propyl-NH-Cbz
(R)-Propyl-NH-Cbz
Propyl-NH-Cbz
Propyl-NH-Cbz
H
R2
R3
H
H
H
H
H
H
H
H
H
H
CH3
CH3
R4
Compound Yield (%) Compound Yield (%)
1
2
3
4
5
6
7
8
9
Isopropyl
Isopropyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Isopropyl
Isopropyl
Isopropyl
H
OtBu
OtBu
OtBu
OMe
OtBu
OtBu
OtBu
OtBu
OtBu
OtBu
OtBu
OMe
2a
2b
2c
2d
2e
2f
2g
2h
2i
94
98
91
77
98
98
99
73
80
83
85
82
3a
3b
3c
3c
3e
3f
3g
3h
3g
3j
86b
99b
95b
99b
99b
96b
99b
93b
73b
99b
84c
99c
Benzyl
Isopropyl
H
Benzyl
Isopropyl
Benzyl
10
11
12
2j
2k
2l
3k
3k
Benzyl
H
a Reactions performed in a monomode microwave CEM Discover apparatus; b Crude yield (pure by NMR); c
Isolated yield after chromatography .
Data in Table 2 clearly shows that the cyclization step assisted by microwave heating yielded DKP
compounds 3a-3k in excellent yields. Because N-Boc protecting groups are unstable at temperatures
higher than 90 °C, microwave irradiation should deprotect the amines facilitating the spontaneous
intramolecular aminolysis [29]. Nature and size of alkyl group (R1) on Cα of Nα-Boc amino acid
residue have no effect on the course and reaction yield when the C terminus amino acid is
phenylalanine (entries 3-7). However, they do influence the cyclization yield when the amino acid at C
terminus is valine (entries 1, 8-10). When valine is present, the amino acid residue sequence is
important on the cyclization yield (entries 7 and 9) because β-branched amino acid at C terminus (Cα
isopropyl group) exerts a bulky effect on ring closure, diminishing the reaction yield (99 vs 73%).
Entries 1 and 3 where a benzyl group replaced an isopropyl one corroborate this steric effect as the
reaction yield diminished from 95 to 86%. The leaving groups OtBu and OMe (ester group nature,
entries 3,4 and 11,12) have no effect on course and reaction yield, as corroborated by the excellent
yields obtained for compounds 3a-3h and 3j-3k. Optical rotation values of compounds 3a-3k and their
comparison with literature data showed that cyclization reaction proceeded without Cα chiral center
epimerization of the amino acid residues. The cyclization of different Nα-Boc-dipeptidyl esters (even
Ot-Bu) in water assisted by microwaves is a rapid, secure, environmentally friendly and highly efficient
method to produce cis-DKPs with high optical purity. This reaction conditions are compatible with the

Molecules 2009, 14
2840
presence of Cbz protecting groups. This protocol was used to obtain the trans-DKP fragment 3b of
compound 1, which was synthesized in quantitative yield starting of Boc-D-Orn(Cbz)-Val-OtBu
(2b, entry 2).
The structures of compounds 2a-2l and 3a-3k were established on the basis of the analysis of their
spectroscopic data. For compounds 2a-2l, carbamate, amide, and ester groups showed IR absorptions
at 3,364-3,283, 2,979-2,972 and 1,690-1,665, and 1,744-1,712 cm-1, respectively; Carbonyl amide,
ester, N-Boc-carbamate, and Nδ-Cbz-carbamate groups showed 13C-NMR resonance signals at
δ 172.35-169.07, δ 172.10-167.89, δ 157.09-155.07, and δ 156.98-156.94. Carbamate and amide
groups in compounds 3a-3k produce IR absorptions at 3,440-3,426, and 2,977-2,925 and 1,678-
13
1,664 cm-1, respectively. Amide groups showed C-NMR resonance signals at δ 167.83-166.00, and
Nδ-Cbz-carbamate carbonyls give resonance signals in the δ 156.06-155.79 range.
3. Experimental
3.1. General procedures
Reactions were performed in sealed vessels in a monomode microwave CEM Discover apparatus
and the temperature was evaluated by infrared. Melting points were obtained in a Fisher Johns melting
point apparatus and are uncorrected. Infrared (IR) spectra were obtained in KBr on a Bruker Vector 22
IR spectrometer. Optical rotations were measured with sodium light (unless otherwise specified) on a
1
13
Perkin-Elmer 341 MC polarimeter. H- and C-NMR spectra were recorded on a Varian Unity 400
1
1
13
spectrometer at 400 MHz for H-NMR, H-1H COSY, HSQC, and HMBC, and at 100 MHz for C-
NMR, using CDCl3 or DMSO as solvents, as indicated. Chemical shifts are reported in ppm (δ)
relative to the TMS signal. FAB+MS, and HRFAB+MS were recorded on a JEOL JMStation-JM 700
mass spectrometer at 70 eV in a matrix of glycerol. Flash column chromatography (FCC) and
analytical thin-layer chromatography (TLC) were performed using silica gel 230-400 mesh and
pre-coated silica gel 60 F254 Merck plates, respectively. Boc-Phe-OH, Boc-Val-OH, Boc-Orn(Cbz)-
OH, Boc-Gly-OH, Sar-OMe, Sar-OtBu, Phe-OMe, Phe-OtBu, Val-OtBu, EDAC, TEA, and DMAP
were obtained from Aldrich, HOBt was obtained from ANASPEC, and all chemicals were used
without further purification.
3.2. General procedure for the syntheses of dipeptides 2a-2j
A mixture of Boc-amino acid (1 mmol), amino ester hydrochloride (1 mmol), EDAC (1.5 mmol),
HOBt (1 mmol) and DMAP (0.1 mmol) were dissolved in dry CH2Cl2 (10 mL). Mixture was cooled to
5 °C and then TEA (1 mmol) was added. Reaction was stirred at 5 °C for further 30 min, then allowed
to warm up to room temperature and stirred overnight. Reaction mixture was treated with sat. NH4Cl
soln. (20 mL). The organic phase was separated and the aqueous layer was extracted with CH2Cl2
(3 × 15 mL). Combined organic layers were washed with brine (2 × 15 mL) and with water
(2 ×15 mL) and dried over Na2SO4. Solvent was removed under vacuum. The residue was purified by
FCC.

Molecules 2009, 14
2841
3.3. General procedure for the syntheses of dipeptides 2k-2l
A mixture of Boc-amino acid (2.4 mmol), amino ester hydrochloride (2 mmol), EDAC (2.4 mmol),
HOBt (2.4 mmol) and DMAP (0.1 mmol) were dissolved in dry CH2Cl2 (5 mL). Mixture was cooled to
5 °C and then TEA (2.4 mmol) was added. Reaction was stirred at 5 °C for further 30 min, then
allowed to warm up to room temperature and stirred for two days. Reaction mixture was treated with
sat. NH4Cl soln. (20 mL). The organic phase was separated and the aqueous layer was extracted with
CH2Cl2 (3 × 15 mL). Combined organic layers were washed with brine (2 × 15 mL) and with water
(2 × 15 mL) and dried over Na2SO4. Solvent was removed under vacuum. The residue was purified
by FCC.
Boc-Orn(Cbz)-Val-OtBu (2a): Colorless syrup; [α] + 8.5 (c 1.09, CHCl3); IR: 3,336, 2,973, 2,935,
2,878, 1,712, 1,666, 1,532, 1,454, 1,368, 1,254, 1,162, 1,018 cm-1; 1H-NMR (CDCl3) δ 7.28-7.20 (5H,
m, Ar), 6.85 (1H, bs, NH-Val), 5.29 (1H, bs, NH-Orn), 5.18 (1H, bs, NHδ-Orn), 5.04 (1H, d, J = 12.4
Hz, CH2-Cbz), 5.01 (1H, d, J = 12.4 Hz, CH2-Cbz), 4.33 (1H, dd, J = 9.2, 4.8 Hz, Hα-Val), 4.24 (1H,
bs, Hα-Orn), 3.29 (1H, bs, Hδ-Orn), 3.08 (1H, bd, J = 13.2 Hz, Hδ’-Orn), 2.09 (1H, dh, J = 9.2, 7.2
Hz, Hβ-Val), 1.78 (1H, m, Hβ-Orn), 1.51 (3H, m, Hβ’-Orn, Hγ-Orn), 1.37 (9H, s, CH3Boc), 1.35 (9H,
s, CH3tBu), 0.86 (3H, d, J = 7.2 Hz, Hγ-Val), 0.84 (3H, d, J = 7.2 Hz, Hγ’-Val); 13C-NMR (CDCl3) δ
172.26 (s, CO-Orn), 170.84 (s, CO-Val), 156.97 (s, CO-Cbz), 155.79 (s, CO-Boc), 136.63 (s, Ar),
128.49 (d, Ar), 128.11 (d, Ar), 128.07 (d, Ar), 81.87 (s, C-Boc), 79.89 (s, C-tBu), 66.80 (t, CH2-Cbz),
57.65 (d, Cα-Val), 53.41 (d, Cα-Orn), 40.02 (t, Cδ-Orn), 31.28 (d, Cβ-Val), 30.10 (t, Cβ-Orn), 28.50
and 28.20 (q, CH3-Boc, CH3-tBu), 26.35 (t, Cγ-Orn), 19.17 (q, Cγ-Val), 17.25 (q, Cγ’-Val); FAB+MS
m/z: 522 (53) [M + H]+, 466 (11) [M + H - C4H8]+, 422 (13) [M + H - C5H8O2]+, 414 (15) [M -
C7H7O]+, 366 (100) [M + H - C5H8O2 - C4H8]+, 258 (13), 213 (20), 91 (100) [C7H7]+, 57 (44) [C4H9]+;
HRFAB+MS: observed 522.3176 [M + H]+, (calcd. for C27H44N3O7, 522.3179).
Boc-D-Orn(Cbz)-Val-OtBu (2b): Colorless syrup; [α] + 15.7 (c 1.04, CHCl3); IR: 3,339, 2,973, 2,935,
2,877, 1,712, 1,666, 1,525, 1,456, 1,391, 1,368, 1,254, 1,165, 1,022, 738, 699 cm-1; 1H-NMR (CDCl3)
δ 7.36-7.25 (5H, m, Ar), 6.89 (1H, bs, NH-Val), 5.36 (1H, bd, J = 6.6 Hz, NH-Orn), 5.25 (1H, bs,
NHδ-Orn), 5.08 (2H, s, CH2-Cbz), 4.39 (1H, dd, J = 8.8, 4.4 Hz, Hα-Val), 4.23 (1H, bs, Hα-Orn), 3.21
(2H, m, Hδ-Orn), 2.10 (1H, dh, J = 9.2, 7.2 Hz, Hβ-Val), 1.85 (1H, m, Hβ-Orn), 1.60 (3H, m, Hβ’-
Orn, Hγ-Orn), 1.45 (9H, s, CH3Boc), 1.43 (9H, s, CH3tBu), 0.92 (3H, d, J = 7.0 Hz, Hγ-Val), 0.89 (3H,
d, J = 7.0 Hz, Hγ’-Val); 13C-NMR (CDCl3) δ 172.06 (s, CO-Orn), 170.78 (s, CO-Val), 156.74 (s, CO-
Cbz), 155.71 (s, CO-Boc), 136.69 (s, Ar), 128.50 (d, Ar), 128.06 (d, Ar), 82.00 (s, C-Boc), 80.06 (s, C-
tBu), 66.68 (t, CH2-Cbz), 57.60 (d, Cα-Val), 54.09 (d, Cα-Orn), 40.37 (t, Cδ-Orn), 31.46 (d, Cβ-Val),
30.07 (t, Cβ-Orn), 28.47 and 28.19 (q, CH3-tBu, CH3-tBu), 26.39 (t, Cγ-Orn), 19.11 (q, Cγ-Val), 17.77
(q, Cγ’-Val); FAB+MS m/z: 522 (9) [M + H]+, 466 (3) [M + H - C4H8]+, 422 (4) [M + H - C5H8O2]+,
410 (9), 366 (39) [M + H - C9H17O2]+, 258 (8), 213 (15), 91 (100) [C7H7]+, 72 (28) [C4H8O] +, 57 (38)
[C4H9]+; HRFAB+MS: observed 522.3176 [M + H]+, (calcd. for C27H44N3O7, 522.3179).
Boc-Orn(Cbz)-Phe-OtBu (2c): White solid; Mp 104-105 °C {lit [30] 102 °C}; [α] + 25.8 (c 1.01,
CHCl3); IR: 3,364, 2,977, 2,880, 1,732, 1,690, 1,668, 1,524, 1,452, 1,368, 1,280, 1,240, 1,164, 1,027
1
cm-1; H-NMR (CDCl3): δ 7.35-7.15 (10H, m, Ar), 6.82 (1H, d, J = 6.8 Hz, NH-Phe), 5.18 (1H, d,

Molecules 2009, 14
2842
J = 6.8 Hz, NH-Orn), 5.06 (2H, d, J = 12.8 Hz, CH2-Cbz, NHδ-Orn), 5.02 (1H, d, J = 12.8 Hz, CH2-
Cbz), 4.70 (1H, dd, J = 14.0, 6.0 Hz, Hα-Phe), 4.24 (1H, bs, Hα-Orn), 3.33 (1H, bs, Hδ-Orn), 3.13
(1H, m, Hδ’-Orn), 3.08 (1H, dd, J = 13.6, 14.0 Hz, Hβ-Phe), 3.04 (1H, dd, J = 13.6, 6.0 Hz, Hβ’-Phe),
2.14 (1H, m, Hγ-Orn), 1.81 (1H, m, Hγ’-Orn), 1.53 (2H, m, Hβ, Hβ’-Orn), 1.43 (9H, s, CH3tBu), 1.37
(9H, s, CH3Boc); 13C-NMR (CDCl3): δ 171.80 (s, CO-Orn), 170.48 (s, CO-Phe), 156.94 (s, CO-Cbz),
155.66 (s, CO-Boc), 136.61 (s, Ar-Cbz), 136.23 (s, Ar), 129.58 (d, Ar), 128.58 (d, Ar), 128.49 (d, Ar),
128.16 (d, Ar), 127.03 (d, Ar), 82.41 (s, C-Boc), 80.07 (s, C-tBu), 66.88 (t, CH2-Cbz), 53.81 (d, Cα-
Phe), 53.35 (d, Cα-Orn), 39.97 (t, Cδ-Orn), 38.16 (t, Cβ-Phe), 30.25 (t, Cγ-Orn), 28.44 and 28.04 (q,
CH3-tBu, CH3-tBu), 26.27 (t, Cβ-Orn); FAB+MS m/z: 570 (56) [M + H]+, 556 (9) [M + H - CH2]+, 514
(8) [M + H - C4H8]+, 470 (56) [M + H - C5H8O2]+, 414 (90) [M + H - C5H8O2 - C4H8]+, 306 (12) [M +
H - C14H19NO3 - CH3]+, 261 (17), 204 (14), 154 (27), 120 (27), 91 (100) [C7H7]+, 57 (44) [C4H9]+;
HRFAB+MS: observed 570.3152 [M + H]+, (calcd. for C31H44N3O7, 570.3179).
Boc-Orn(Cbz)-Phe-OMe (2d): White solid; Mp 118-120 °C {lit [31] 106-118 °C}; [α] - 7.5 (c 1.01,
MeOH) {lit [31] - 7.6 (c 1.1, MeOH)}; IR: 3,339, 2,974, 2,939, 2,876, 1,743, 1,677, 1,531, 1,449,
1
1,369, 1,278, 1,249, 1,172, 1,032 cm-1; H-NMR (CDCl3) δ 7.36-7.10 (10H, m, Ar), 7.00 (1H, d,
J = 7.6 Hz, NH-Phe), 5.24 (1H, d, J = 8.0 Hz, NH-Orn), 5.12 (1H, t, J = 6.0 Hz, NHδ-Orn), 5.03 (1H,
d, J = 12.8 Hz, CH2-Cbz), 4.99 (1H, d, J = 12.8 Hz, CH2-Cbz), 4.83 (1H, dd, J = 13.2, 6.4 Hz, Hα-
Phe), 4.25 (1H, bs, Hα-Orn), 3.67 (3H, s, OCH3), 3.33 (1H, m, Hδ-Orn), 3.12 (1H, m, Hδ’-Orn), 3.11
(1H, dd, J = 13.6, 5.6 Hz, Hβ-Phe), 3.05 (1H, dd, J = 13.6, 6.8 Hz, Hβ’-Phe), 1.78 (1H, m, Hγ-Orn),
1.52 (3H, m, Hβ, Hβ’, Hγ-Orn), 1.42 (9H, s, CH3Boc); 13C-NMR (CDCl3) δ 172.10 (s, CO-Phe),
171.91 (s, CO-Orn), 156.98 (s, CO-Cbz), 155.68 (s, CO-Boc), 136.58 (s, Ar-Cbz), 135.95 (s, Ar),
129.31 (d, Ar), 128.64 (d, Ar), 128.55 (d, Ar), 128.14 (d, Ar), 127.14 (d, Ar), 80.04 (s, C-Boc), 66.82
(t, CH2-Cbz), 53.48 (d, Cα-Phe), 53.28 (d, Cα-Orn), 52.47 (q, OCH3), 39.95 (t, Cδ-Orn), 38.05 (t, Cβ-
Phe), 30.26 (t, Cγ-Orn), 28.52 (q, CH3Boc), 26.24 (t, Cβ-Orn); FAB+MS m/z: 528 (11) [M + H]+, 472
(7) [M + H - C4H9]+, 428 (62) [M + H - C5H8O2]+, 320 (9) [C17H24N2O4]+, 275(10), 180 (19)
[C10H14NO2]+, 120 (31) [C8H10N]+, 91 (100) [C7H7]+, 57 (44) [C4H9]+; HRFAB+MS: observed
528.2686 [M + H]+, (calcd. for C28H38N3O7, 528.2710).
Boc-Gly-Phe-OtBu (2e): Colorless syrup; [α] + 47.2 (c 1.1, CHCl3); IR: 3,414, 3,336, 2,979, 2,934,
1
1,727, 1,671, 1,521, 1,452, 1,369, 1,220, 1,160, 1,044, 942, 850, 743, 701 cm-1; H-NMR (CDCl3): δ
7.30-7.13 (5H, m, Ar), 6.72 (1H, d, J = 8.0 Hz, NH-Phe), 5.32 (1H, dd, J = 4.8, 4.8 Hz, NH-Gly), 4.75
(1H, dd, J = 14.0, 6.0 Hz, Hα-Phe), 3.83 (1H, dd, J = 16.4, 5.2 Hz, Hα-Gly), 3.74 (1H, dd, J = 16.4,
5.2 Hz, Hα’-Gly), 3.08 (2H, d, J = 6.0 Hz, Hβ-Phe), 1.44 (9H, s, CH3-tBu), 1.39 (9H, s, CH3-Boc);
13C-NMR (CDCl3) δ 170.39 (s, CO-Phe), 169.07 (s, CO-Gly), 155.97 (s, CO-tBu), 136.63 (s, Ar),
129.54 (d, Ar), 128.43 (d, Ar), 127.00 (d, Ar), 82.54 (s, C-Boc), 80.21 (s, C-tBu), 53.66 (d, Cα-Phe),
44.32 (t, Cα-Gly), 38.22 (t, Cβ-Phe), 28.47 and 28.09 (q, CH3-tBu, CH3-tBu); FAB+MS m/z: 379 (41)
[M + H]+, 323 (16) [M + H - C4H8]+, 267 (100) [M + H - C4H8 - C4H8]+, 223 (25) [M + H -
C7H10NO3]+, 166 (13), 154 (57), 120 (28), 57 (38) [C4H9]+; HRFAB+MS: observed 379.2267 [M +
H]+, (calcd. for C20H31N2O5, 379.2233).

Molecules 2009, 14
2843
1
Boc-Phe-Phe-OtBu (2f): Colorless crystals; Mp 125-127 °C; [α] + 34.1 (c 1.0, CHCl3); IR, H-NMR
13
and C-NMR (CDCl3) are in agreement with previously reported data [32]; FAB+MS m/z: 469 (27)
[M + H]+, 413 (10) [M + H - C4H8]+, 357 (58) [M + H - C4H8 - C4H8]+, 313 (78) [M + H - C4H8 -
C5H8O2]+, 166 (15), 120 (100), 57 (45) [C4H9]+; HRFAB+MS: observed 469.2724 [M + H]+, (calcd. for
C27H37N2O5, 469.2702).
Boc-Val-Phe-OtBu (2g): Colorless crystals; Mp 112-115 °C; [α] + 29.0 (c 1.01, CHCl3); IR: 3,337,
3,282, 2,972, 2,936, 2,874, 1,733, 1,689, 1,659, 1,525, 1,457, 1,371, 1,248, 1,162, 1,020, 848, 752, 696
1
cm-1; H-NMR (CDCl3): δ 7.30-7.15 (5H, m, Ar), 6.50 (1H, d, J = 7.2 Hz, NH-Phe), 5.16 (1H, d,
J = 8.8 Hz, NH-Val), 4.74 (1H, dd, J = 14.0, 6.4 Hz, Hα-Phe), 3.94 (1H, dd, J = 8.4, 6.8Hz, Hα-Val),
3.08 (2H, dd, J = 6.4, 4.4 Hz, Hβ-Phe), 2.09 (1H, m, Hβ-Val), 1.45 (9H, s, CH3-tBu), 1.38 (9H, s,
13
CH3-Boc), 0.93 (3H, d, J = 6.8 Hz, Hγ-Val), 0.88 (3H, d, J = 6.4 Hz, Hγ’-Val); C-NMR (CDCl3) δ
171.16 (s, CO-Val), 170.46 (s, CO-Phe), 155.79 (s, CO-Boc), 136.10 (s, Ar), 129.57 (d, Ar), 128.45 (d,
Ar), 127.02 (d, C4), 82.42 (s, C-Boc), 79.89 (s, C-tBu), 60.03 (d, Cα-Val), 53.78 (d, Cα-Phe), 38.34 (t,
Cβ-Phe), 31.16 (d, Cβ-Val), 28.52 and 28.09 (q, CH3-tBu, CH3-Boc), 19.44 (q, Cγ-Val), 17.97 (q, Cγ’-
Val); FAB+MS m/z: 421 (59) [M + H]+, 365 (17) [M + H - C4H8]+, 309 (100) [M + H - C4H8 - C4H8]+,
265 (95) [M + H - C4H8 - C5H8O2]+, 166 (30), 120 (60), 72 (53), 57 (48) [C4H9]+; HRFAB+MS:
observed 421.2666 [M + H]+, (calcd. for C23H37N2O5, 421.2702).
Boc-Gly-Val-OtBu (2h): Colorless oil; [α] + 23.8 (c 1.02, CHCl3); IR: 3,333, 2,975, 2,935, 2,878,
1
1,726, 1,671, 1,524, 1,458, 1,391, 1,369, 1,281, 1,251, 1,166, 1,052, 943, 848, 786 cm-1; H-NMR
(CDCl3) δ 6.80 (1H, bs, NH-Val), 5.48 (1H, bd, J = 5.6 Hz, NH-Gly), 4.46 (1H, dd, J = 9.2, 4.4 Hz,
Hα-Val), 3.87 (1H, dd, J = 16.4, 5.6 Hz, Hα-Gly), 3.80 (1H, dd, J = 16.4, 5.6 Hz, Hα’-Gly), 2.17 (1H,
hd, J = 7.2, 4.4 Hβ-Val), 1.47 (9H, s, CH3Boc), 1.46 (9H, s, CH3tBu), 0.94 (3H, d, J = 7.2 Hz, Hγ-
Val), 0.89 (3H, d, J = 7.2 Hz, Hγ’-Val); 13C-NMR (CDCl3) δ 170.95 (s, CO-Val), 169.52 (s, CO-Gly),
156.12 (s, CO-Boc), 82.16 (s, C-Boc), 80.22 (s, C-tBu), 57.42 (d, Cα-Val), 44.50 (t, Cα-Gly), 31.58
(d, Cβ-Val), 28.46 and 28.20 (q, CH3-Boc, CH3-tBu), 19.08 (q, Cγ-Val), 17.68 (q, Cγ’-Val); FAB+MS
m/z: 331 (28) [M + H]+, 275 (18) [M + H - C4H8]+, 219 (100) [M + H - C4H8 - C4H8]+, 175 (20) [M +
H - C4H8 - C5H8O2]+, 72 (17) [C4H8O]+, 57 (20) [C4H9]+; HRFAB+MS: observed 331.2251 [M + H]+,
(calcd. for C16H31N2O5, 331.2233).
Boc-Phe-Val-OtBu (2i): White solid; Mp 119-121 °C; [α]DHg (365 nm) + 9.0 (c 0.5, CHCl3); IR: 3,327,
1
2,975, 2,933, 1,735, 1,687, 1,651, 1,538, 1,367, 1,252, 1,165, 1,025, 855 cm-1; H-NMR (CDCl3) δ
7.32 - 7.10 (5H, Ar), 6.56 (1H, d, J = 8.4 Hz, NH-Phe), 5.20 (1H, d, J = 8.0 Hz, NH-Val), 4.41 (1H, m,
Hα-Phe), 4.36 (1H, dd, J = 8.0, 4.8 Hz, Hα-Val), 3.10 (2H, dd, J = 13.6, 6.4 Hz, Hβ-Phe), 3.04 (1H,
dd, J = 13.6, 6.8 Hz, Hβ’-Phe), 2.17 (1H, hd, J = 6.8, 4.8 Hβ-Val), 1.45 (9H, s, CH3Boc), 1.41 (9H, s,
13
CH3tBu), 0.88 (3H, d, J = 6.8 Hz, Hγ-Val), 0.89 (3H, d, J = 6.8 Hz, Hγ’-Val); C-NMR (CDCl3) δ
171.07 (s, CO-Phe), 170.45 (s, CO-Val), 155.39 (s, CO-Boc), 136.69 (s, C1), 129.36 (d, C2 and C6),
128.58 (d, C3 and C5), 126.85 (d, C4), 81.98 (s, C-Boc), 80.10 (s, C-tBu), 57.65 (d, Cα-Val), 55.95 (d,
Cα-Phe), 38.23 (t, Cβ-Phe), 31.62 (d, Cβ-Val), 28.43 and 28.26 (q, CH3-Boc, CH3-tBu), 18.92 (q, Cγ-
Val), 17.91 (q, Cγ’-Val); FAB+MS m/z: 421 (46) [M + H]+, 365 (15) [M + H - C4H8]+, 309 (100) [M +

Molecules 2009, 14
2844
H - C4H8 - C4H8]+, 265 (96) [M + H - C4H8 - C5H8O2]+, 120 (40), 72 (32) [C4H8O]+, 57 (44) [C4H9]+;
HRFAB+MS: observed 421.2688 [M + H]+, (calcd. for C23H37N2O5, 421.2702).
Boc-Val-Val-OtBu (2j): White solid; Mp 132-134 °C; [α] - 6.8 (c 1.1, CHCl3); IR: 3,309, 2,972, 2,934,
2,888, 1,744, 1,685, 1,651, 1,536, 1,464, 1,372, 1,301, 1,254, 1,219, 1,157, 1,017, 855 cm-1; 1H-NMR
(CDCl3) δ 6.35 (1H, d, J = 7.6 Hz, NH-Val), 5.09 (1H, d, J = 8.8 Hz, NH-Val), 4.36 (1H, dd, J = 8.4,
4.4 Hz, Hα-Val), 3.86 (1H, dd, J = 7.6, 7.6 Hz, Hα-Val), 2.08 (2H, m, Hβ-Val, Hβ-Val), 1.40 (9H, s,
CH3Boc), 1.38 (9H, s, CH3tBu), 0.90, 0.87, 0.86, 0.84 (3H each, d, J = 6.8 Hz, Hγ-Val, Hγ’-Val, Hγ-
Val, Hγ’-Val); 13C-NMR (CDCl3) δ 171.58 (s, CO-Val), 170.81 (s, CO-Val), 155.93 (s, CO-Boc),
81.16 (s, C-Boc), 80.00 (s, C-tBu), 60.38 (d, Cα-Val), 57.70 (d, Cα-Val), 31.64 (d, Cβ-Val), 31.06 (d,
Cβ-Val), 28.56 and 28.29 (q, CH3-Boc, CH3-tBu), 19.57, 19.16, 18.21, 17.96 (q, Cγ-Val, Cγ’-Val, Cγ-
Val, Cγ’-Val); FAB+MS m/z: 373 (46) [M + H]+, 317 (28) [M + H - C4H8]+, 261 (89) [M + H - C4H8 -
C4H8]+, 217 (94) [M + H - C4H8 - C5H8O2]+, 116 (26) [C5H10NO2]+, 72 (100) [C4H8O]+, 57 (47)
[C4H9]+; HRFAB+MS: observed 373.2711 [M + H]+, (calcd. for C19H37N2O5, 373.2702).
Boc-Phe-Sar-OtBu (2k): Colorless syrup (73:27 rotamer mixture); [α] - 22.5 (c 1.58, CHCl3);
IR: 3,427, 3,322, 2,978, 2,933, 1,741, 1,710, 1,652, 1,491, 1,367, 1,236, 1,164, 1,049, 1,020, 952, 850,
759 and 701 cm-1; 1H-NMR (CDCl3) δ 7.30-7.00 (5H, m, Ar), 5.31 (1H, d, J = 8.8 Hz, NH-Phe), 4.80
(1H, dd, J = 15.4, 6.6 Hz, Hα-Phe), 3.92 (1H, d, J = 17.2 Hz, Hα-Sar), 3.84 (1H, d, J = 17.2 Hz, Hα’-
Sar), 2.83 (3H, s, NCH3-Sar), 2.97 (1H, dd, J = 13.6, 7.2 Hz, Hβ-Phe), 2.90-2.86 (1H, m, Hβ’-Phe),
13
1.38 (9H, s, CH3-Boc), 1.31 (9H, s, CH3-tBu); C-NMR (CDCl3) δ 172.11 (s, CO-Phe), 167.89 (s,
CO-Sar), 155.09 (s, CO-Boc), 136.42 (s, C1), 129.65 (d, C2,C6), 128.38 (d, C3,C5), 126.85 (d, C4),
82.03 (s, C-tBu), 79.67 (s, C-Boc), 51.52 (d, Cα-Phe), 50.45 (t, Cα-Sar), 39.57 (t, Cβ-Phe), 36.34 (q,
NCH3-Sar), 28.49 and 28.24 (q, CH3-Boc, CH3-tBu); FAB+MS m/z: 393 (27) [M + H]+, 337 (15) [M +
H - C4H8]+, 281 (76) [M + H - 2 C4H8]+, 263 (15) [M + H - C4H8 - C4H8O]+, 237 (100) [M + H - C4H8
- C5H8O2]+, 164 (18), 120 (81), 90 (43) [C7H6]+, 57 (78) [C4H9]+; HRFAB+MS: observed 393.2409 [M
+ H]+, (calcd. for C21H33N2O5, 393.2389).
Boc-Phe-Sar-OMe (2l): Colorless syrup (77:23 rotamer mixture); [α] + 19.1 (c 0.54, CHCl3); IR:
3,427, 3,322, 2,977, 2,943, 1,751, 1,708, 1,652, 1,490, 1,407, 1,365, 1,250, 1,212, 1,171, 1,047, 1,021,
751 and 702 cm-1; 1H-NMR (CDCl3) δ 7.32-7.18 (5H, m, Ar), 5.34 (1H, d, J = 8.8 Hz, NH-Phe), 4.88
(1H, dd, J = 14.8, 6.8 Hz, Hα-Phe), 4.14 (1H, d, J = 17.2 Hz, Hα-Sar), 3.99 (1H, d, J = 17.2 Hz, Hα'-
Sar), 3.73 (3H, s, OCH3), 3.04 (1H, dd, J = 13.6, 7.2 Hz, Hβ-Phe), 2.95 (1H, dd, J = 13.6, 6.4 Hz, Hβ’-
Phe), 2.87 (3H, s, NCH3-Sar), 1.40 (9H, s, CH3-Boc); 13C-NMR (CDCl3) δ 172.35 (s, CO-Phe), 169.28
(s, CO-Sar), 155.07 (s, CO-Boc), 136.33 (s, Ar), 129.65 (d, Ar), 128.45 (d, Ar), 126.93 (d, C4), 79.81
(s, C-Boc), 52.38 (q, OCH3), 51.59 (d, Cα-Phe), 49.66 (t, Cα-Sar), 39.80 (t, Cβ-Phe), 36.43 (q, NCH3-
Sar), 28.55 (q, CH3-Boc); FAB+MS m/z: 351 (33) [M + H]+, 295 (55) [M + H - C4H8]+, 251 (100) [M +
H - C5H9O2]+, 164 (16), 120 (67), 104 (70) [C4H10NO2]+, 57 (44) [C4H9]+, 44 (18); HRFAB+MS:
observed 351.1907 [M + H]+, (calcd. for C18H27N2O5, 351.1920).

Molecules 2009, 14
2845
3.4. General procedure for the syntheses of 2,5-diketopiperazines 3a-3k
Each Nα-Boc-dipeptidyl ester (0.25 mmol) was dissolved or suspended in water (1 mL) and heated
during 10 minutes at 250 °C and 150 psi, using a monomode CEM Discover microwave apparatus at
250 W. The resulting suspension was filtered through a Hirsch funnel and washed with water (5 mL),
the solid was dried under high vacuum and analyzed without further purification by NMR. Compounds
3h and 3k were water soluble, and in these cases, resulting solutions were lyophilized and the solids
purified as indicated in Table 2 and analyzed by NMR.
Cyclo[Val-Orn(Cbz)] (3a): White solid; Mp 202-204 °C {lit [33] 206-208 °C}; [α] - 26.0 (c 0.27,
DMSO) {lit [33] - 47.4 (c 1 %)}; IR: 3,434, 3,333, 3,201, 3,094, 3,052, 2,965, 2,878, 1,678, 1,531,
1,444, 1,258, 1,142, 1,025, 774, 696, 629 cm-1; 1H-NMR (DMSO): δ 8.14 (1H, s, NH-Orn), 8.04 (1H,
s, NH-Val), 7.39-7.26 (6H, m, Ar, NHδ-Orn), 5.00 (2H, s, CH2-Cbz), 3.81 (1H, t, J = 4.8 Hz, Hα-Orn),
3.66 (1H, bs, Hα-Val), 2.97 (1H, dd, J = 12.8, 6.4 Hz, Hδ-Orn), 2.14 (1H, m, Hβ-Val), 1.69 (1H, m,
Hβ-Orn), 1.62 (1H, m, Hβ’-Orn), 1.46 (2H, m, Hγ-Orn), 0.93 (3H, d, J = 7.2 Hz, Hγ-Val), 0.82 (3H,
m, d, J = 7.2 Hz, Hγ’-Val); 13C-NMR (DMSO): δ 167.83 (s, CO-Orn), 166.88 (s, CO-Val), 156.06 (s,
CO-Cbz), 137.21 (s, Ar), 128.33 (d, Ar), 127.64 (d, Ar), 65.18 (t, CH2-Cbz), 59.43 (d, Cα-Val), 53.75
(d, Cα-Orn), 40.18 (t, Cδ-Orn), 31.33 (d, Cβ-Val), 31.16 (t, Cβ-Orn), 25.35 (t, Cγ-Orn), 18.74 (q, Cγ-
Val), 17.29 (q, Cγ’-Val); FAB+MS m/z: 348 (87) [M + H]+, 307 (100) [M + H - C3H6]+, 289 (61), 240
(22) [M + H - C7H8O]+, 219 (25) [C12H15N2O2]+, 214 (12), 195 (12), 165 (12); HRFAB+MS: observed
348.1887 [M + H]+, (calcd. for C18H26N3O4, 348.1923).
Cyclo[Val-D-Orn(Cbz)] (3b): White solid; Mp 212-214 °C; [α] + 11.7 (c 0.5, MeOH); IR: 3,347,
3,192, 3,054, 2,962, 1,675, 1,540, 1,460, 1,265, 1,143, 1,031, 852, 696 cm-1; 1H-NMR (DMSO): δ 8.11
(1H, s, NH-Orn), 7.32 (6H, m, Ar, NH-Val), 4.99 (2H, s, CH2-Cbz), 4.03 (1H, bs, Hα-Val), 3.88 (1H,
bt, Hα-Orn), 2.96 (1H, d, J = 5.6 Hz, Hδ-Orn), 2.09 (1H, m, Hβ-Val), 1.65 (1H, m, Hβ-Orn), 1.62 (1H,
m, Hβ’-Orn), 1.41 (2H, m, Hγ-Orn), 0.92 (3H, d, J = 6.8 Hz, Hγ-Val), 0.83 (3H, m, d, J = 7.0 Hz, Hγ’-
13
Val); C-NMR (DMSO): δ 168.13 (s, CO-Orn), 167.67 (s, CO-Val), 156.17 (s, CO-Cbz), 137.25 (s,
Ar), 128.44 (d, Ar), 127.77 (d, Ar), 65.30 (t, CH2-Cbz), 59.79 (d, Cα-Val), 53.27 (d, Cα-Orn), 40.18
(t, Cδ-Orn), 32.19 (d, Cβ-Val), 29.64 (t, Cβ-Orn), 24.41 (t, Cγ-Orn), 18.54 (q, Cγ-Val), 17.08 (q, Cγ’-
Val); FAB+MS m/z: 348 (3) [M + H]+, 219 (5), 154 (12), 130 (30), 107 (8), 91 (100), 85 (28), 72 (25);
HRFAB+MS: observed 348.1949 [M + H]+, (calcd. for C18H26N3O4, 348.1923).
Cyclo[Phe-Orn(Cbz)] (3c): White solid; Mp 200-202 °C {lit [33] 210-212 °C}; [α] - 24.8 (c 0.24,
DMSO) {lit [33] - 12.9 (c 1 %)}; IR: 3,322, 3,189, 3,039, 2,965, 2,894, 1,669, 1,532, 1,458, 1,338,
1,246, 1,134, 1,101, 1,016, 852, 753, 669 cm-1; 1H-NMR (DMSO): δ 8.16 (1H, s, NH-Phe), 8.05 (1H,
s, NH-Orn), 7.40 - 7.05 (10H, m, Ar), 5.00 (2H, s, CH2-Cbz), 4.17 (1H, s, Hα-Phe), 3.57 (1H, bs, Hα-
Orn), 3.13 (1H, dd, J = 13.6, 4.0 Hz, Hβ-Phe), 2.83 (1H, dd, J = 13.6, 5.2 Hz, Hβ’-Phe), 2.68 (1H, dd,
J = 12.8, 6.4 Hz, Hδ-Orn), 1.04 (1H, m, Hβ-Orn), 0.85 (2H, m, Hγ-Orn), 0.65 (2H, m, Hβ’-Orn); 13C-
NMR (DMSO): δ 166.70 (s, CO-Orn), 166.00 (s, CO-Phe), 155.79 (s, CO-Cbz), 137.16 (s, Ar-Cbz),
135.92 (s, Ar), 130.16 (d, Ar), 128.27 (d, Ar), 127.92 (d, Ar), 127.69 (d, Ar), 126.57 (d, Ar), 65.10 (t,
CH2-Cbz), 55.30 (d, Cα-Phe), 53.61 (d, Cα-Orn), 39.95 (t, Cδ-Orn), 38.16 (t, Cβ-Phe), 30.66 (t, Cβ-
Orn), 24.33 (t, Cγ-Orn); FAB+MS m/z: 396 (100) [M + H]+, 352 (32) [M + H - H2NCO]+, 335 (9), 307

Molecules 2009, 14
2846
(53) [M + H - C7H6]+, 289 (32) [M + H - C7H7O]+, 243 (16) [M + H - C14H16NO2]+, 219 (18) [M + H -
C10H12NO2]+, 165 (14); HRFAB+MS: observed 396.1949 [M + H]+, (calcd. for C22H26N3O4,
396.1923).
Cyclo(Phe-Gly) (3e): White solid; Mp 266-268 °C {lit [34] 271-273 °C }; [α] + 26.6 (c 0.95, DMSO)
{lit [34] + 7.3 (c 0.95, DMSO)}; IR: 3,426, 3,190, 3,057, 2,977, 2,921, 2,878, 1,676, 1,462, 1,332,
1
13
1,086, 1,004, 847, 794, 758, 702 cm-1; H- and CNMR (DMSO) are in agreement with previously
reported data [35]; FAB+MS m/z: 205 (13) [M + H]+, 169 (15), 154 (24), 130 (43) [M + H - C4H4]+, 85
(100) [C3H3NO2]+; HRFAB+MS: observed 205.1058 [M + H]+, (calcd. for C11H13N2O2, 205.0977).
1
Cyclo(Phe-Phe) (3f): White solid; [α] - 42.8 (c 0.2, AcOH); H- and 13C-NMR (DMSO), and
HRFAB+MS are in agreement with previously reported data [36]; IR: 3,440, 3,318, 3,198, 3,056,
2,970, 2,929, 1,669, 1,458, 1,338, 1,197, 1,088, 1,013, 759 and 700 cm-1.
Cyclo(Val-Phe) (3g): White solid; Mp 264-266 °C {lit [36] 263-265 °C}; [α] - 66.0 (c 0.28, DMSO)
{lit [37] - 64 (c 0.2 AcOH); [34] - 43.3 (c 0.27 DMSO)}; IR: 3,440, 3,316, 3,192, 3,056, 2,967, 2,885,
1
1,668, 1,454, 1,341, 1,090, 859, 758, 699, cm-1; H- and 13C-NMR (DMSO) are in agreement with
previously reported data [34,37]; FAB+MS m/z: 247 (100) [M + H]+, 219 (13) [M + H - CO]+, 217
(10), 203 (7), 165 (5); HRFAB+MS: observed 247.1446 [M + H]+, (calcd. for C14H19N2O2, 247.1447).
Cyclo(Val-Gly) (3h): White solid; Mp 210-212 °C {lit [38] 256 °C}; [α] + 32.9 (c 0.46, H2O) {lit [38]
+ 23.7 (c 1.0, H2O)}; IR: 3,431, 3,199, 3,055, 2,925, 2,859, 1,670, 1,461, 1,381, 1,346, 1,109, 1,047,
1
808, 621 cm-1; H- and 13C-NMR (DMSO) are in agreement with previously reported data [38,39];
FAB+MS m/z: 157 (94) [M + H]+, 154 (100) [M - H2], 136 (78), 120 (12), 107 (25), 85 (58) [C5H9O]+,
77 (25), 55 (32), 43 (27) [C3H7]+, 41 (25); HRFAB+MS: observed 157.0957 [M + H]+, (calcd. for
C7H13N2O2, 157.0957).
Cyclo(Val-Val) (3j): Colorless needless; Mp 268 °C with sublimation {lit [40] 268 °C}; [α] - 54.8 (c
0.5, AcOH) {lit [15] - 62 (c 0.5, AcOH); IR: 3,434, 3,325, 3,193, 3,100, 3,057, 2,966, 2,880, 1,664,
1,449, 1,346, 1,293, 847 cm-1; 1H-NMR (DMSO): δ 7.96 (1H, s, NH-Val), 3.69 (1H, s, Hα-Val), 2.18
(1H, m, Hβ-Val), 0.95 (3H, d, J = 7.6 Hz, Hγ-Val), 0.83 (3H, d, J = 7.6 Hz, Hγ’-Val); 13C-NMR
(DMSO): δ 167.26 (s, CO), 59.09 (d, Cα), 31.06 (d, Cβ), 18.73 (q, Cγ), 17.33 (q, Cγ’); FAB+MS m/z:
199 (100) [M + H]+, 197 (19), 169 (8); HRFAB+MS: observed 199.1463 [M + H]+, (calcd. for
C10H19N2O2, 199.1447).
Cyclo(Sar-Phe) (3k): Colorless needles; Mp 180-183 °C {lit [41] 184-185 °C}; [α] + 56.0 (c 1.01,
MeOH) {lit [41] + 47.5 (c 2.3 MeOH)}; IR: 3,440, 3,253, 2,963, 2,929, 2,895, 1,656, 1,470, 1,441,
1
1,322, 1,102, 1,033, 870, 754, 699, cm-1; H-NMR (CDCl3): δ 7.60 (1H, bs, NH-Phe), 7.35-7.16 (5H,
m, Ar), 4.31 (1H, bs, Hα-Phe), 3.48 (1H, d, J = 17.6 Hz, Hα-Sar), 3.24 (1H, dd, J = 13.6, 5.2 Hz, Hβ-
Phe), 3.07 (1H, dd, J = 13.6, 4.4 Hz, Hβ’-Phe), 2.81 (3H, s, NCH3-Sar), 2.79 (1H, d, J = 17.6 Hz, Hα’-
Sar); 13C-NMR (CDCl3): δ 166.32 (s, CO-Sar), 165.52 (s, CO-Phe), 134.99 (s, Ar), 130.07 (d, Ar),
128.63 (d, Ar), 127.58 (d, C4), 56.53 (d, Cα-Phe), 50.92 (t, Cα-Sar), 40.94 (t, Cβ-Phe), 33.66 (s,

Molecules 2009, 14
2847
NCH3-Sar); FAB+MS m/z: 219 (100) [M + H]+, 154 (50) [M + H - CO]+, 136 (56), 91 (35) [C7H7]+, 73
(58); HRFAB+MS: observed 219.1134 [M + H]+, (calcd. for C12H15N2O2, 219.1134).
4. Conclusions
Optically pure cis-DKPs could be synthesized in one-pot from the corresponding Nα-Boc-
dipeptidyl-tert-butyl esters in water under microwave irradiation for ten minutes. Employing these
conditions, the tert-butoxy group is efficiently removed, leading to cyclization in excellent yields. This
is the first protocol for ring closures using Nα-Boc-dipeptidyl-tert-butyl esters. The trans-DKP
fragment present in the natural product 1 was synthesized in quantitative yield. The reaction is rapid,
secure, environmentally friendly and highly efficient.
Acknowledgements
This work was financially supported by CONACyT (Grants number 79584-Q and 2006-C01-LN-
56431). Lemuel Pérez-Picaso thanks CONACyT for a doctoral fellowship (number 181754). We are
grateful to Dr. A. Berenice Aguilar-Guadarrama, Dr. Blanca Domínguez Mendoza, Dr. Diana Gabriela
Vargas Pineda, Ing. Victoria Labastida, and T. C. María Medina Pastor for technical assistance.
References and Notes
1. Wipf, P. Synthetic studies of biologically active marine cyclopeptides. Chem. Rev. 1995, 95,
2115-2134.
2. Amidon, G.L.; Lee, H.J. Absorption of peptide and peptidomimetic drugs. Annu. Rev. Pharmacol.
Toxicol. 1994, 34, 321-341.
3. Liu, S.; Gu, W.; Lo, D.; Ding, X.; Ujiki, M.; Adrian, T.E.; Soff, G.A.; Silverman, R.B.
N-Methylsansalvamide A peptide analogues. Potent new antitumor agents. J. Med. Chem. 2005,
48, 3630-3638.
4. Prasad, C. Bioctive cyclic dipeptides. Peptides 1995, 16, 151-164.
5. Houston, D.R.; Synstad, B.; Eijsink, V.G.H.; Stark, M.J.R.; Eggleston, I.M.; van Aalten, D.M.F.
Structure-based exploration of cyclic dipeptide chitinase inhibitors. J. Med. Chem. 2004, 47,
5713-5720.
6. Martins, M.B.; Carvalho, I. Diketopiperazines: biological activity and synthesis. Tetrahedron
2007, 63, 9923-9932.
7. Graz, C.J.M.; Grant, G.D.; Brauns, S.C.; Hunt, A.; Jamie, H.; Milne, P.J. Cyclic dipeptides in the
induction of maturation for cancer therapy. J. Pharm. Pharmacol. 2000, 52, 75-82.
8. Prakash, K.R.C.; Tang, Y.; Kozikowski, A.P.; Flippen-Anderson, J.L.; Knoblach, S.M.; Faden,
A.I. Synthesis and biological activity of novel neuroprotective diketopiperazines. Bioorg. Med.
Chem. 2002, 10, 3043-3048.
9. Lambert, J.N.; Mitchell, J.P.; Roberts, K.D. The synthesis of cyclic peptides. J. Chem. Soc. Perkin
Trans. I 2001, 471-484.
10. Rajappa, S.; Natekar, M.V. Piperazine-2,5-diones and related lactam ethers. Adv. Het. Chem.
1993, 57, 187-289.

Molecules 2009, 14
2848
11. Fischer, P.M.J. Diketopiperazines in peptide and combinatorial chemistry. J. Pept. Sci. 2003, 9,
9-35.
12. Dinsmore, C.J.; Beshore, D.C. Recent advances in the synthesis of diketopiperazines. Tetrahedron
2002, 58, 3297-3312.
13. Rodionov, I.L.; Rodionova, L.N.; Baidakova, L.K.; Romashko, A.M.; Balashova, T.A.; Ivanov,
V.T. Cyclic dipeptides as building blocks for combinatorial libraries. Part 2: Synthesis of
bifunctional diketopiperazines. Tetrahedron 2002, 58, 8515-8523.
14. Fischer, E. Synthese von polypeptiden. Chem. Ber. 1906, 39, 2893-2931.
15. Ueda, T.; Saito, M.; Kato, T.; Izumiya, N. Facile Synthesis of Cyclic Dipeptides and Detection of
Racemization. Bull. Chem. Soc. Jpn. 1983, 56, 568-572.
16. Suzuki, K.; Sasaki, Y.; Endo, N.; Mihara, Y. Acetic acid-catalized diketopiperazine synthesis.
Chem. Pharm. Bull. 1981, 29, 233-237.
17. Nitecki, D.E.; Halpern, B.; Westley, J.W. Simple route to sterically pure dioxopiperazines. J. Org.
Chem. 1968, 33, 864-866.
18. Eriksson, J.; Arvidsson, P.I.; Davidsson, O. Solution structure of a dilithiumamide/diethylzinc
heterocomplex that catalyzes asymmetric alkylation reactions. Chem. Eur. J. 1999, 5, 2356-2361.
19. Lee, S.; Kanmera, T.; Aoyagi, H.; Izumiya, N. Cyclic peptides. VI. Asymmetric hydrogenation of
dehydroalanine or dehydroaminobutanoic acid residue in cyclodipeptides. Int. J. Pept. Protein
Res. 1979, 13, 207-217.
20. Kappe, C.O.; Dallinger, D. Controlled microwave heating in modern organic synthesis: highlights
from the 2004-2008 literature. Mol. Divers. 2009, 13, 71-193.
21. Kappe, C.O.; Dallinger, D.; Murphree, S.S. Practical microwave synthesis for organic chemists:
Strategies, Instruments, and Protocols, 1st ed.; Wiley-VCH: Darmstadt, Germany, 2009.
22. Santagada, V., Fiorino, F.; Perissutti, E.; Severino, B.; Terracciano, S.; Cirino, G.; Caliendo, G. A
convenient strategy of dimerization by microwave heating and using 2,5-diketopiperazine as
scaffold. Tetrahedron Lett. 2003, 44, 1145-1148.
23. López-Cobeñas, A.; Cledera, P.; Sánchez, J.D.; Pérez-Contreras, R.; López-Alvarado, P.; Ramos,
M.T.; Avendaño, C.; Menéndez, J.C. Solvent-Free, Efficient Synthesis of 2,5-Piperazinediones
from Boc-protected dipeptide esters under microwave irradiation. Synlett 2005, 7, 1158-1160.
24. Tullberg, M.; Grøtli, M.; Luthman, K. Efficient synthesis of 2,5-diketopiperazines using
microwave assisted heating. Tetrahedron 2006, 62, 7484-7491.
25. Jam, F.; Tullberg, M.; Luthman, K.; Grøtli, M. Microwave assisted synthesis of spiro-2,5-
diketopiperazines. Tetrahedron 2007, 63, 9881-9889.
26. Fischer, P.M.; Solbakken, M.; Undheim, K. Solution synthesis of a dimeric pentapeptide:
diketopiperazine cyclisation of Glu-Asp dipeptide esters and Asp-racemisation during segment
condensation. Tetrahedron 1994, 50, 2277-2288.
27. Besser, D.; Greiner, G.; Reissmann, S. Side reaction with N-carboxymethyl amino acids in the
synthesis of lactone cyclized peptides. Lett. Pept. Sci. 1998, 5, 299-303.
28. Pérez-Picaso, L.; Rios, M.Y.; Hernández, A.N.; Martínez, J. 1H and 13C assignments of cyclo [N-
(Lys-Phe)-Orn-Val], a semicyclic imide tetrapeptide from Burkholderia cepacia. Magn. Reson.
Chem. 2006, 44, 959-961.

Molecules 2009, 14
2849
29. Pandey, S.K.; Awasthi, K.K.; Saxena, A.K. Microwave assisted stereospecific synthesis of
(S)-3-substituted 2,3,6,7,12,12a-hexahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-diones.
Tetrahedron 2001, 57, 4437-4432.
30. Kobayashi, N.; Higuchi, T.; Urano, Y.; Kikuchi, K.; Hirobe, M.; Nagano, T. Dipeptides
Containing L-arginine analogs: New isozyme-selective inhibitors of nitric oxide synthase. Biol.
Pharm. Bull. 1999, 22, 936-940.
31. Shen, H.Y.; Tian, G.L.; Ye, Y.H.; Wang, J. Non-coded amino acids as acyl donor substrates for
peptide bond formation catalyzed by thermoase in toluene. J. Mol. Catal. B-Enzym. 2005, 37,
26-29.
32. Dineen, T.A.; Zajac, M.A.; Myers, A.G. Efficient transamidation of primary carboxamides by in
situ activation with N,N-dialkylformamide dimethyl acetals. J. Am. Chem. Soc. 2006, 128,
16406-16409.
33. Izumiya, N.; Kato, T.; Fujita, Y.; Ohno, M.; Kondo, M. Studies of peptide antibiotics, I. Dipeptide
anhydrides as models of cyclic peptide antibiotics. Bull. Chem. Soc. Jpn. 1964, 37, 1809-1816.
34. López-Cobeñas, A.; Cledera, P.; Sánchez, J.D.; López-Alvarado, P.; Ramos, M.T.; Avendaño, C.;
Menéndez, J.C. Microwave-assisted synthesis of 2,5-piperazinediones under solvent-free
conditions. Synthesis 2005, 19, 3412-3422.
35. Huang, H.; She, Z.; Lin, Y.; Vrijmoed, L.L.P.; Lin, W. Cyclic Peptides from an Endophytic
Fungus Obtained from a Mangrove Leaf (Kandelia candel). J. Nat. Prod. 2007, 70, 1696-1699.
36. Joshi, K.B., Verma, S. Participation of aromatic side chains in diketopiperazine ensembles.
Tetrahedron Lett. 2008, 49, 4231-4234.
37. Tullberg, M.; Luthman, K.; Grøtli, M. Microwave-assisted solid-phase synthesis of 2,5-
diketopiperazines: Solvent and resin dependence. J. Comb. Chem. 2006, 8, 915-922.
38. Bull, S.D.; Davies, S.G.; Moss, W.O. Practical synthesis of Schöllkopf's bis-lactim ether chiral
auxiliary: (3S)-3,6-dihydro-2,5-dimethoxy-3-isopropyl-pyrazine. Tetrahedron: Asym. 1998, 9,
321-327.
39. Cledera, P.; Avendaño, C.; Menéndez, J.C. Comparative study of synthetic approaches to 1-
arylmethylenepyrazino[2,1-b]quinazoline-3,6-diones. Tetrahedron 1998, 54, 12349-12360.
40. Tanihara, M.; Hiza, T.; Imanishi, Y.; Higashimura, T. Solution conformation of cyclic dipeptides
having aliphatic side chains. Bull. Chem. Soc. Jpn. 1983, 56, 1155-1160.
41. Lucente, G.; Pinnen, F.; Zanotti, G. Cyclization of activated N-benzyloxycarbonyl-tripeptides.
Tetrahedron Lett. 1978, 11, 1009-1012.
Sample Availability: Samples of the compounds 2a-2l and 3a-3k are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).