One-Pot ugi/aza-michael synthesis of highly substituted 2,5-diketopiperazines with anti-proliferative properties

Article abstract of DOI:10.3390/molecules171214685

The well-known Ugi reaction of aldehydes with amines, carboxylic acids and isocyanides leads to the formation of acyclic a-acylaminocarboxamides. Replacement of the carboxylic acid derivatives with β-acyl substituted acrylic acids gives access to highly substituted 2,5-diketopiperazines in one single reaction-step without additives or complex reaction procedures. The obtained diketopiperazines show anti-proliferative effects on activated T cells and represent therefore potential candidates for targeting unwanted T cell-mediated immune responses.

Full text of DOI:10.3390/molecules171214685

Molecules 2012, 17, 14685-14699; doi:10.3390/molecules171214685
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
One-Pot Ugi/Aza-Michael Synthesis of Highly Substituted
2,5-Diketopiperazines with Anti-Proliferative Properties
Andreas Hartung 1, Florian Seufert 1, Carsten Berges 2, Viktoria H. Gessner 3 and
Ulrike Holzgrabe 1,*
1
Institute of Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland,
97074 Wuerzburg, Germany; E-Mails: andreas.hartung@uni-wuerzburg.de (A.H.);
f.seufert@pharmazie.uni-wuerzburg.de (F.S.)
2
Department of Internal Medicine II, Translational Immunotherapy, University Hospital Wuerzburg,
Versbacherstr 5, 97078 Wuerzburg, Germany; E-Mail: berges_c@medizin.uni-wuerzburg.de
3
Institute of Inorganic Chemistry, University of Wuerzburg, Am Hubland, 97074 Wuerzburg,
Germany; E-Mail: vgessner@uni-wuerzburg.de
* Author to whom correspondence should be addressed;
E-Mail: u.holzgrabe@pharmazie.uni-wuerzburg.de; Tel.: +49-931-31-85460;
Fax: +49-931-31-85494.
Received: 21 November 2012; in revised form: 1 December 2012 / Accepted: 6 December 2012 /
Published: 11 December 2012
Abstract: The well-known Ugi reaction of aldehydes with amines, carboxylic acids and
isocyanides leads to the formation of acyclic α-acylaminocarboxamides. Replacement of
the carboxylic acid derivatives with β-acyl substituted acrylic acids gives access to highly
substituted 2,5-diketopiperazines in one single reaction-step without additives or complex
reaction procedures. The obtained diketopiperazines show anti-proliferative effects
on activated T cells and represent therefore potential candidates for targeting unwanted
T cell-mediated immune responses.
Keywords: multicomponent Ugi-type reaction; intramolecular Michael addition;
2,5-diketopiperazines; anti-proliferative effects

Molecules 2012, 17
14686
1. Introduction
Nowadays, multicomponent reactions (MCRs) are part of the conventional repertoire of synthetic
chemists for a couple of good reasons. In contrast to a sequential multi-step approach, MCRs usually
give more or less complex product molecules out of three or more starting materials in one single step.
In consequence the reaction times and the unavoidable efforts on the reaction and the workup
procedure are significantly reduced. However, the most important advantage of multicomponent
reactions in academic and industrial research is their variability [1]. By consecutively changing the
structures of the starting compounds, a diverse library of numerous compounds can be created very
quickly. As this feature correlates perfectly with the demands of medicinal chemistry, MCRs are
notably utilized in lead structure optimization. E.g., starting from xylocaine [2], which is an important
local anaesthetic and accessible within just one step, numerous analogue compounds have been
synthesized in the same way and are now commercially available anaesthetic drugs [1,3].
In an effort to synthesize a smart library of Ugi products with potential antitumoral activity
(unpublished results) we started off with the well-known Ugi four-component (U-4CR) reaction using
acrylic acids, to obtain highly diverse Michael systems 5 [4]. However, introducing β-acyl substituted
acrylic acid derivatives influences the reaction outcome and gives access to the pharmacological
attractive scaffold of 2,5-piperazindiones 6 (2,5-DKPs) [5–7], without any heating or microwave
irradiation (see Figure 1). Preliminary pharmacological investigations suggest the synthesized DKPs
are promising candidates for drugs with anti-proliferative activity.
Figure 1. The U-4CR reaction with α,β-unsaturated carboxylic acids gives the expected
Ugi product 5, whereas the usage of β-acyl substituted acrylic acid gives rise to highly
substituted 2,5-diketopiperazines 6.
2. Results and Discussion
While highly substituted 2,5-DKPs are conventionally synthesized via classic dipeptide formation [8,9]
or dipeptide synthesis via the Ugi reaction [7,10], both followed by a deprotection and cyclization step,
a direct one-pot synthesis pathway has recently attracted attention [11]. An aldehyde [12] or keto

Molecules 2012, 17
14687
compound [13] can be converted with an amine, isocyanide and β-acyl substituted acrylic acid under
microwave irradiation necessary to promote an Aza-Michael ring closure. It was reported that this
reaction can be applied to various alkyl- and benzyl isocyanides which are starting materials known to
give reasonable yields in the Ugi reaction.
Although the use of p-toluenesulfonylmethyl isocyanide (TosMIc) is more discerning due to its low
reactivity [14,15], we decided to extent the scope of the reaction in this way for two reasons: (1) highly
lipophilic compounds tend to address many targets unspecifically and increase promiscuity [16] and
(2) compounds need to be water soluble for in vitro and in vivo studies. TosMIc therefore is highly
appropriate as it provides a polar and metabolic fairly inert sulfone function. Here we report the
synthesis of a couple of structurally different tosyl substituted 2,5-DKPs with anti-proliferative properties
according to the elegant multicomponent pathway by thermal means or even without any heating.
The reaction of 2-bromobenzyaldehyde (1a), 3-(aminomethyl)pyridine (2a), TosMIc (3) and
cinnamic acid (4a) gave the desired acyclic Ugi-product 5 as expected (Figure 1). However, replacing
the carboxylic acid component by 3-benzoylacrylic acid (4b–d)–which just differs in one carbonyl
group–resulted in the ring closed 2,5-diketopiperazine product 6a (Figure 1, Table 1). The key step is
an intra-molecular aza-1,4-Michael addition, classified as a 6-exo-trig reaction. In contrast, compound
5 and several analogues (not shown), also containing a Michael-system, do not undergo the 7-endo-trig
reaction to form the corresponding 7-membered bislactam ring under comparable conditions (Figure 1).
Hence, the essential structural moiety for the cyclisation reaction is the double Michael system of the
butenedione, which can be introduced by any β-acyl substituted acrylic acid derivative.
Table 1. Synthesized acyclic Ugi product 5 and 2,5-diketopiperazines 6a–j.
Yield
#
1
2
3
4
5
6
R1
R2
R3
R4
product cis:trans
[%]
pyridin-
3ylmethyl (2a)
pyridin-
2-Br-Ph (1a)
2-Br-Ph (1a)
Ph (1b)
tosylmethyl (3)
tosylmethyl (3)
tosylmethyl (3)
tosylmethyl (3)
tosylmethyl (3)
tosylmethyl (3)
Ph (4a)
(±) 5
(±) 6a
(±) 6b
(±) 6c
(±) 6d
(±) 6e
-
17
benzoyl (4b)
benzoyl (4b)
benzoyl (4b)
benzoyl (4b)
benzoyl (4b)
1.6:1
trans a
2.2:1
1:1
35 b
45
3ylmethyl (2a)
pyridin-
3ylmethyl (2a)
pyridin-
2-Cl-Ph (1c)
2-I-Ph (1d)
4-F-Ph (1e)
35 b
43 b
42
3ylmethyl (2a)
pyridin-
3ylmethyl (2a)
pyridin-
trans a
3ylmethyl (2a)

Molecules 2012, 17
14688
Table 1. Cont.
Yield
[%]
55
#
R1
R2
R3
R4
product cis:trans
7
8
9
Ph (1b)
Ph (1b)
Ph (1b)
Bn (2b)
phenethyl (2c)
4-MeO-Bn (2d)
pyridin-
tosylmethyl (3)
benzoyl (4b)
benzoyl (4b)
benzoyl (4b)
4-Cl-benzoyl
(4c)
(±) 6f
(±) 6g
(±) 6h
trans a
trans a
trans a
tosylmethyl (3)
tosylmethyl (3)
12
39
10
11
Ph (1b)
tosylmethyl (3)
(±) 6i
trans a
37
36
3ylmethyl (2a)
pyridin-
4-MeO-
Ph (1b)
tosylmethyl (3)
(±) 6j
trans a
3ylmethyl (2a)
benzoyl (4d)
a Precipitation of the pure trans product; b Yields of the purely isolated diastereomeric mixture is reported.
In order to investigate the initially observed anti-proliferative properties in more detail, a small
series of different derivatives was synthesised (Table 1). It is noteworthy that all reactions were carried
out between room temperature and 55 °C by thermal heating. In contrast to a recently published work
mainly using fumaric acid in the synthesis of 2,5-DKPs [12], no microwave irradiation was necessary
for the considered reactions. Moreover, the syntheses of the products reported here could not take
advantage of being carried out under the influence of microwaves or of using water as solvent. The
moderate yields obtained are in similar range as reported for related multicomponent reactions
employing TosMIc [17].
The X-ray crystal structure of 6b (Figure 2) not only confirmed the structure deduced from NMR
spectroscopy, but also provided information about the relative configuration. In accordance to the
NMR analyses, the substituents at C-3 and C-6 of the almost flat DKP ring are in a trans-
configuration. When trans-oriented, the dipolar coupling mediated proton-proton correlation–known as
Nuclear Overhauser Effect (NOE)–was observed between the proton at C-3 and the methylene protons
attached to C-6 (Figure 2, correlation I) and/or between the proton at C-6 and the ortho-protons of the
phenyl substituent at C-3 (Figure 2, correlation II). In contrast, when the substituents are in the
cis-position, a NOE correlation was observed between the methylene protons at the substituent of C-6
and the ortho-protons of the phenyl ring at C-3 (Figure 2, correlation III) [18]. While the majority of
the synthesized compounds precipitated readily out of the reaction mixtures, compounds 6a, 6c and 6d
needed purification by means of column chromatography. Interestingly, by precipitation only one of
the two possible diastereomers was isolated. Moreover, the filtrate of two representatively investigated
reaction mixtures (compounds 6b and 6j) did not contain any cis-isomer. The synthesis of compounds
6a, 6c and 6d gave cis:trans diastereomeric ratios ranging from 1:1 to 1:2 (determined using the
integrals of the NMR signals of the hydrogens attached to C-3) and were separated by means of double
column chromatography.

Molecules 2012, 17
14689
Figure 2. The X-ray crystal structure of 6b shows the trans substitution at C-3 and C-6 of
the DKP (left). NOESY correlations indicate the relative configuration at the DKP core.
I, II: trans; III: cis (right).
2,5-DKPs have been shown to exhibit anticancer activity by inhibiting proliferation of cancer cell
lines [19,20]. In order to analyze the anti-proliferative effect of 2,5-DKPs we used activated T cells
because T cells are not only key cells for the initiation of an adaptive immune response, but also
participate in the onset of dysregulated immune responses like inflammatory diseases, autoimmune
diseases and transplant rejection [21,22]. Thus we aimed to clarify if 2,5-DKPs can also be used for
treatment of these unwanted T cell-mediated immune responses. For comparative purposes, heat shock
protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) which
selectively blocks proliferation of activated T cells, was included in the assay [23].
T cells activated in a physiological manner by allogeneic dendritic cells (DC) were incubated for
24 h with the synthesized compounds, 17-DMAG and DMSO control and subsequently proliferation of
activated T cells was determined by [3H]-thymidine uptake. As shown in Figure 3, the trans
compounds 6a, 6c, 6d and 6i reduced significantly proliferation of activated T cells in a dose-
dependent manner, confirming results of other studies which showed that 2,5-DKPs are able to
suppress proliferation of highly proliferating cells of different origin [19,20].
High molecular concentrations (50 µM) of the 2,5-trans-DKPs 6a, 6c, 6d and 6i suppressed
proliferation of activated T cells more intensively than the highest used 17-DMAG concentration
(10 µM). However, this molecular concentration of 17-DMAG were more effective in inhibiting
proliferation of activated T cells than equal concentrations of the trans compounds 6a, 6c and 6d. In
contrast, compounds 6b, 6e, 6f, 6g and 6h and the cis compounds 6a, 6c and 6d showed a lower or no
anti-proliferative effect on activated T cells (Supplementary Table 1).

Molecules 2012, 17
14690
Figure 3. Purified human T cells were activated by coculture with allogeneic human DCs
for 5 days. Activated human T cells were exposed for the last 24 h of coculture to the
indicated concentrations [µM] of synthesized trans compounds (white bars), cis
compounds (black bars), 17-DMAG (red bars) or DMSO ctrl, and incorporation of
[3H]thymidine was determined as described in the Experimental section. Data are given as
mean values ± standard error of the mean of four independent experiments carried out in
triplicate. * p < 0.05, ** p < 0.01 compared to DMSO control.
Analysing the structure-activity relationships of the synthesized compounds, bromo and chloro
substitution in R1 proved to be favourable (Br ≈ Cl > I > H > F). With regard to the influence of R2, the
polar pyridin-3ylmethyl and MeO-benzyl substituents are privileged over the lipophilic benzyl
and phenethyl groups. Variations in R4 demonstrate a preference for the chlorobenzoyl residue
(Cl-benzoyl > benzoyl > MeO-benzoyl).
To investigate the cytotoxicity we incubated resting T cells for 24 h with increasing concentrations
of compounds 6a–j, 17-DMAG and DMSO control and afterwards measured apoptosis induction by
defining percentage of Annexin V+/7-AAD+ cells. We noticed that incubation of resting T cells with
all used concentrations of compound 6d (trans), 6e and 6f caused no apoptosis, whereas high
molecular concentrations of compound 6a (cis and trans), 6c (cis and trans) and 6d (cis) induced
negligible apoptosis which is comparable to apoptosis rate induced by DMAG. In contrast, all used
concentrations of compound 6g induced a moderate level and of compound 6i even a considerable
level of apoptosis in resting T cells (see Supplementary Table 1). Thus, the observed anti-proliferative
effect on activated T cells of compound 6g and 6i can be most likely attributed to cytotoxic activities.
To summarize, trans-compounds 6a, 6c and 6d significantly inhibited proliferation of activated T
cells and showed at high molecular concentrations an even more profound anti-proliferative effect
than highest used 17-DMAG concentration, combined with negligible cytotoxicity. Drugs of this
pharmacological profile are urgently needed.

Molecules 2012, 17
14691
3. Experimental
3.1. General
Melting points were determined by means of a Stuart melting point apparatus SMP11 (Bibby
Scientific, Stone, UK) and not corrected. IR spectra were obtained using a JASCO FT/IR-6100
spectrometer (JASCO, Gross-Umstadt, Germany). TLC was performed on silica gel 60 F254-coated
1
13
glass sheets (Merck, Darmstadt, Germany). H (400.132 MHz) and C (100.613 MHz) NMR spectra
were recorded on a Bruker AV 400 instrument (Bruker Biospin, Ettlingen, Germany). As internal
1
13
standard the signals of the deuterated solvent was used (DMSO-d6: H 2.50 ppm, C 39.52 ppm;
1
13
CDCl3: H 7.26 ppm, C 77.16 ppm). Abbreviations for data quoted are: s, singlet; d, doublet; t,
triplet; m, multiplet; b, broad; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet
of triplets. Coupling constants (J) are given in Hz. LCMS data were obtained using a Agilent 1100
Series LC/MSD Trap (Agilent Technologies, Böblingen, Germany). HRMS data were obtained using a
Bruker micrOTOF focus II instrument (Bruker Daltonik, Bremen, Germany). The elemental analyses
(CHN) were recorded on a EURO EA 3000 Elemental Analyzer instrument (Eurovektor Instruments,
Hekatech, Germany). Chemicals were purchased from Aldrich (Steinheim, Germany) and Thermo
Fisher Scientific (Geel, Belgium) and used without purification.
3.2. Procedure for the Synthesis of the Acyclic Ugi-Product
N-(1-(2-Bromophenyl)-2-oxo-2-((tosylmethyl)amino)ethyl)-N-(pyridine-3-ylmethyl)cinnamamide (5).
To a solution of 2-bromobenzaldehyde (3 mmol, 350 µL) in absolute methanol (5 mL)
3-(aminomethyl)pyridine (3 mmol, 310 µL) was added and stirred for 20 min at room temperature.
Then p-toluenesulfonyl-methyl isocyanide (3.6 mmol, 700 mg) and cinnamic acid (3 mmol, 450 mg)
were added. After 96 h the solvent was evaporated, the residue dissolved in CHCl3 and extracted with
a saturated aqueous solution of NaHCO3. The organic layer was dried over Na2SO4 and evaporated.
The crude product was purified by means of column chromatography (silica gel 60, toluene/ethyl
acetate 1:3) to give the pure 5. Colorless amorphous solid (310 mg, 17%): mp 184–186 °C; IR ν 2952,
1689, 1643, 1594, 1415, 1320, 1288, 1138, 816, 762, 704 cm−1; 1H-NMR (400 MHz, CHCl3) δ 8.31 (d,
3.4 Hz, 1H), 8.10 (s, 1H), 7.85 (d, 15.3 Hz, 1H), 7.71 (d, 8.0 Hz, 2H), 7.51 (br. s, 1H), 7.40 (br. s, 3H),
7.36–7.31 (m, 2H), 7.27 (d, 8.0 Hz, 2H), 7.36–7.01 (m, 4H), 7.22–7.14 (m, 1H), 7.11–7.00 (m, 1H),
6.69 (d, 15.3 Hz, 1H), 6.54 (s, 1H), 4.91 (dd, 13.7 Hz, 7.3 Hz, 1H), 4.80 (d, 18.0 Hz, 1H), 4.63–4.49
(m, 2H), 2.39 (s, 3H); 13C NMR (100 M Hz, CHCl3): δ 169.6, 167.8, 147.7, 147.1, 145.6, 145.2, 134.6,
134.5, 134.0, 133.2, 132.9, 132.7, 131.3, 130.8, 130.2, 130.0, 128.9, 128.6, 128.2, 128.1, 127.8, 123.2,
116.4, 61.2, 60.3, 46.8, 21.7; LCMS (ESI+): m/z 619.5 [M+H]+; Anal. calcd. for C31H28BrN3O4S: C
60.20, H 4.56, N 6.79, S 5.18; found: C 60.04, H 4.60, N 6.88, S 5.01.
3.3. General Procedure for the Synthesis of Piperazine-2,5-diones 6a–j
To a solution of benzaldehyde derivative (1 eq.) in absolute methanol the corresponding amine
(1 eq.) was added and stirred for 30 min at room temperature. Then p-toluenesulfonylmethyl
isocyanide (1 eq.) and the corresponding benzoylacryclic acid derivative (1 eq.) were added. The

Molecules 2012, 17
14692
solution was stirred until the reaction, controlled by means of TLC, was finished. If necessary, the
reaction mixture was heated.
3-(2-Bromophenyl)-6-(2-oxo-2-phenylethyl)-4-(pyridin-3-ylmethyl)-1-(tosylmethyl)piperazine-2,5-dione
(6a). Quantities: 2-bromobenzaldehyde (470 µL, 4.0 mmol), 3-(aminomethyl)pyridine (410 µL,
4.0 mmol), p-toluenesulfonylmethyl isocyanide (940 mg, 4.8 mmol), 3-benzoylacrylic acid (710 mg,
4.0 mmol). The solvent was evaporated in vacuo and the residue was purified by column
chromatography (1st silica gel 60, n-hexane/ethyl acetate 1:20) to give the purified mixture of cis- and
trans-product in a yield of 35%. A second purification step by column chromatography (2nd flash
silica gel, dichloromethane/methanol 10:0.5) gave the cis- and trans-diastereomers separately. trans-6a:
Colorless amorphous solid (341 mg, 13%): mp 111–114 °C; IR ν 1657, 1596, 1422, 1319, 1140, 1085,
1
762, 688 cm−1; H-NMR (400 MHz, DMSO) δ 8.40 (dd, 4.8 Hz, 1.6 Hz, 1H), 8.06 (d, 1.8 Hz, 1H),
8.02 (dd, 7.8 Hz, 1.2 Hz, 2H), 7.71 (d, 8.1 Hz, 2H), 7.70 (tt, 7.8 Hz, 1.2 Hz, 1H), 7.58 (t, 7.8 Hz, 2H),
7.55 (dd, 7.7 Hz, 1.3 Hz, 1H), 7.37–7.29 (m, 2H), 7.35 (d, 8.1 Hz, 2H), 7.26 (td, 7.7 Hz, 1.7 Hz, 1H),
7.23 (dd, 7.8 Hz, 4.8 Hz, 1H), 7.13 (dd, 7.7 Hz, 1.7 Hz, 1H), 5.68 (s, 1H), 5.36 (d, 14.8 Hz, 1H), 4.97
(d, 14.8 Hz, 1H), 4.96–4.92 (m, 1H), 4.84 (d, 15.4 Hz, 1H), 4.33 (dd, 19.1 Hz, 1H), 3.92 (dd, 19.1 Hz,
13
1H), 3.74 (d, 15.4 Hz, 1H), 2.34 (s, 3H); C-NMR (100 MHz, DMSO): δ 197.6, 165.9, 164.5, 149.1,
148.4, 144.7, 135.8, 135.6, 135.2, 135.0, 133.7, 132.8, 130.9, 130.6, 130.3, 129.8, 128.6, 128.1 (2C),
128.0, 124.1, 123.1, 62.8, 62.4, 55.9, 45.0, 39.9, 21.0; HRMS (MS-TOF): [M+H]+ calcd. for
C32H29BrN3O5S: 646.1006, found: 646.1001. cis-6a: Colorless amorphous solid (161 mg, 6%): mp
1
99–100 °C; IR ν 1657, 1595, 1422, 1323, 1140, 1084, 761, 688 cm-1; H-NMR (400 MHz, DMSO) δ
8.48 (dd, 4.7 Hz, 1.7 Hz, 1H), 8.24 (d, 1.7 Hz, 1H), 8.10 (d, 9.3 Hz, 1.2 Hz, 2H), 7.84 (dd, 7.8 Hz,
1.3 Hz, 1H), 7.70 (tt, 7.4 Hz, 1.2 Hz, 1H), 7.63–7.56 (m, 4H), 7.56 (dd, 7.8 Hz, 1.0 Hz, 1H), 7.51 (dt,
7.9 Hz, 1.7 Hz, 1H), 7.45 (td, 7.8 Hz, 1.0 Hz, 1H), 7.34 (dd, 7.9 Hz, 4.7 Hz, 1H), 7.27 (td, 7.8 Hz, 1.3 Hz,
1H), 7.19 (d, 8.1 Hz, 2H), 5.24 (s, 1H), 5.06–5.00 (m, 1H), 5.12 (d, 14.7 Hz, 1H), 5.01 (d, 14.7 Hz,
1H), 4.87 (d, 15.5 Hz, 1H), 4.21 (dd, 18.7 Hz, 4.4 Hz, 1H), 3.99 (dd, 18.7 Hz, 4.1 Hz, 1H), 3.76 (d,
15.5 Hz, 1H), 2.26 (s, 3H); 13C NMR (100 M Hz, DMSO): δ 196.9, 165.2, 163.9, 149.1, 148.6, 144.7,
136.1, 135.6, 135.0, 134.1, 133.4, 132.7, 131.1, 130.4, 129.8, 129.6, 128.6, 128.4, 128.3, 128.1, 124.7,
123.4, 63.9, 61.4, 56.0, 44.9, 40.6, 21.0; HRMS (MS-TOF): [M+H]+ calcd. for C32H29BrN3O5S:
646.1006, found: 646.1008.
3-(2-Oxo-2-phenylethyl)-6-phenyl-1-(pyridin-3-ylmethyl)-4-(tosylmethyl)piperazine-2,5-dione (trans-6b).
Quantities: benzaldehyde (900 µL, 8.9 mmol), 3-(aminomethyl)pyridine (910 µL, 8.9 mmol),
p-toluenesulfonylmethyl isocyanide (1.74 g, 8.9 mmol), 3-benzoylacrylic acid (1.60 g, 8.9 mmol). The
precipitate was filtered, dissolved in dichloromethane and crystallization initiated by adding diethyl
ether to give pure trans-6b. Colorless solid (2.28 g, 45%): mp 204–205 °C; IR ν 2952, 1662, 1597,
1429, 1418, 1319, 1289, 1141, 1087, 757, 713, 691 cm−1; 1H-NMR (400 MHz, DMSO) δ 8.42 (dd, 4.9 Hz,
1.6 Hz, 1H), 8.17 (d, 1.6 Hz, 1H) 8.00 (d, 7.5 Hz, 2H), 7.69 (t, 7.5 Hz, 1H), 7.63 (d, 8.3 Hz, 2H), 7.57
(t, 7.5 Hz, 2H), 7.41 (dt, 7.5 Hz, 1.6 Hz, 1H), 7.39–7.30 (m, 1H), 7.33 (d, 7.8 Hz, 2H), 7.27 (d, 8.3 Hz,
2H), 7.25 (dd, 7.5 Hz, 4.9 Hz, 1H ), 7.08 (dd, 7.8 Hz, 1.2 Hz, 2H), 5.48 (d, 14.9 Hz, 1H), 5.20 (s, 1H),
4.93 (dd, 4.4 Hz, 3.5 Hz, 1H), 4.88 (d, 14.9 Hz, 1H), 4.86 (d, 15.3 Hz, 1H), 4.23 (dd, 18.9 Hz, 4.4 Hz,
1H), 3.88 (dd, 18.9 Hz, 3.5 Hz, 1H), 3.79 (d, 15.3 Hz, 1H), 2.34 (s, 3H); 13C-NMR (100 MHz, DMSO):

Molecules 2012, 17
14693
δ 197.3, 165.7, 165.5, 149.0, 148.3, 144.7, 135.9, 135.7, 135.4, 134.6, 133.6, 131.4, 129.8, 128.6,
128.5, 128.3, 128.03, 127.97, 127.90, 123.1, 63.4, 61.7, 55.4, 45.2, 37.9, 21.0; LCMS (ESI+):
m/z 567.4 [M]+; Anal. calcd. for C32H29N3O5S: C 67.71, H 5.15, N 7.40, S 5.65; found: C 67.39,
H 5.11, N 7.33, S 5.54.
3-(2-Chlorophenyl)-6-(2-oxo-2-phenylethyl)-4-(pyridin-3-ylmethyl)-1-(tosylmethyl)piperazine-2,5-dione
(6c). Quantities: 2-chlorobenzaldehyde (230 µL, 2.0 mmol), 3-(aminomethyl)pyridine (200 µL,
2.0 mmol), p-toluenesulfonylmethyl isocyanide (390 mg, 2.0 mmol), 3-benzoylacrylic acid (350 mg,
2.0 mmol). The solvent was evaporated in vacuo and the residue was purified by column
chromatography (1st silica gel 60, dichloromethane/methanol 10:0.5) to give the purified mixture of
cis- and trans-product in a yield of 35%. A second purification step by column chromatography (2nd
flash silica gel, chloroform/ethanol 10:0.1) gave the cis- and trans-diastereomers separately. trans-6c:
Slightly yellow solid (102 mg, 9%): mp 135–138 °C; IR ν 2940, 1652, 1596, 1433, 1324, 1141, 1086,
1029, 815, 753, 689 cm−1; 1H-NMR (400 MHz, DMSO) δ 8.40 (dd, 4.7 Hz, 1.7 Hz, 1H), 8.05 (d, 1.7 Hz,
1H), 8.01 (dd, 7.7 Hz, 1.2 Hz, 2H), 7.71 (d, 8.3 Hz, 2H), 7.69 (tt, 7.7 Hz, 1.2 Hz, 1H), 7.57 (t, 7.7 Hz,
2H), 7.38–7.31 (m, 5H), 7.30–7.25 (m, 1H), 7.23 (dd, 7.8 Hz, 4.7 Hz, 1H), 7.17 (d, 7.0 Hz, 1H), 5.66
(s, 1H), 5.38 (d, 14.8 Hz, 1H), 4.97 (d, 14.8 Hz, 1H), 4.94 (dd, 4.2 Hz, 3.4 Hz, 1H), 4.83 (d, 15.4 Hz,
1H), 4.31 (dd, 19.1 Hz, 4.2 Hz, 1H), 3.92 (dd, 19.1 Hz, 3.4 Hz, 1H), 3.77 (d, 15.4 Hz, 1H), 2.34 (s,
13
3H); C-NMR (100 MHz, DMSO): δ 197.6, 165.9, 164.6, 149.1, 148.4, 144.7, 135.8, 135.5, 135.0,
133.7, 133.5, 133.4, 130.9, 130.7, 130.1, 129.8, 129.6, 128.6, 128.07, 128.05, 127.4, 123.1, 62.8, 60.4,
55.9, 45.0, 39.9, 21.0; HRMS (MS-TOF): [M+H]+ calcd. for C32H29ClN3O5S: 602.1511, found:
602.1510. cis-6c: slightly yellow solid (103 mg, 9%): mp 79–82 °C; IR ν 2934, 1657, 1596, 1423,
1326, 1141, 1085, 1028, 815, 752, 688 cm−1; 1H-NMR (400 MHz, DMSO) δ 8.48 (dd, 4.7 Hz, 1.7 Hz,
1H), 8.22 (d, 1.7 Hz, 1H), 8.09 (d, 7.5 Hz, 2H), 7.83 (d, 7.8 Hz, 1H), 7.70 (tt, 7.5 Hz, 1.2 Hz, 1H), 7.60
(d, 8.0 Hz, 2H), 7.59 (t, 7.5 Hz, 2H), 7.51 (dt, 7.9 Hz, 1.7 Hz, 1H), 7.44–7.36 (m, 3H), 7.36–7.32 (m,
1H), 7.18 (d, 8.0 Hz, 2H), 5.23 (s, 1H), 5.12 (d, 14.5 Hz, 1H), 5.03 (dd, 4.4 Hz, 4.2 Hz, 1H), 5.00 (d,
14.5 Hz, 1H), 4.88 (d, 15.4 Hz, 1H), 4.19 (dd, 18.7 Hz, 4.4 Hz, 1H), 3.98 (dd, 18.7 Hz, 4.2 Hz, 1H),
3.77 (d, 15.4 Hz, 1H), 2.26 (s, 3H); 13C NMR (100 MHz, DMSO): δ 196.9, 165.3, 163.9, 149.1, 148.6,
144.7, 136.1, 135.6, 134.1, 133.8, 133.4, 133.2, 131.0, 130.2, 129.8, 129.6, 129.4, 128.6, 128.3, 128.1,
127.8, 123.3, 63.9, 58.9, 56.0, 45.0, 40.7, 20.9; HRMS (MS-TOF): [M+H]+ calcd. for C32H29ClN3O5S:
602.1511, found: 602.1513.
3-(2-Iodophenyl)-6-(2-oxo-2-phenylethyl)-4-(pyridin-3-ylmethyl)-1-(tosylmethyl)piperazine-2,5-dione
(6d). Quantities: 2-iodobenzaldehyde (465 mg, 2.0 mmol), 3-(aminomethyl)pyridine (200 µL,
2.0 mmol), p-toluenesulfonylmethyl isocyanide (390 mg, 2.0 mmol), 3-benzoylacrylic acid (350 mg,
2.0 mmol). The solvent was evaporated in vacuo and the residue was purified by column
chromatography (1st silica gel 60, dichloromethane/methanol 10:0.5) to give the purified mixture of
cis- and trans-product in a yield of 45%. A second purification step by column chromatography (2nd
flash silica gel, chloroform/ethanol 10:0.2) gave the cis- and trans-diastereomers separately. trans-6d:
Colorless amorphous solid (210 mg, 15%): mp 101–104 °C; IR ν 2929, 1657, 1596, 1580, 1422, 1318,
1
1140, 1085, 1013, 815, 759, 688 cm−1; H-NMR (400 MHz, DMSO) δ 8.41 (dd, 4.8 Hz, 1.7 Hz, 1H),
8.08 (d, 1.7 Hz, 1H), 8.02 (dd, 7.9 Hz, 1.3 Hz, 2H), 7.81 (dd, 7.7 Hz, 1.4 Hz, 1H), 7.73–7.67 (m, 2H),
7.71 (d, 8.2 Hz, 2H), 7.58 (t, 7.9 Hz, 2H), 7.35 (d, 8.2 Hz, 2H), 7.32 (td, 7.7 Hz, 1.1 Hz, 1H), 7.25 (dd,

Molecules 2012, 17
14694
7.9 Hz, 4.8 Hz, 1H), 7.07 (td, 7.7 Hz, 1.4 Hz, 1H), 7.03 (dd, 7.7 Hz, 1.1 Hz, 1H), 5.61 (s, 1H), 5.39 (d,
14.7 Hz, 1H), 4.96–4.93 (m, 1H), 4.95 (d, 14.7 Hz, 1H), 4.86 (d, 15.5 Hz, 1H), 4.35 (dd, 19.1 Hz, 4.1 Hz,
1H), 3.91 (dd, 19.1 Hz, 3.2 Hz, 1H), 3.67 (d, 15.5 Hz, 1H), 2.34 (s, 3H); 13C-NMR (100 MHz,
DMSO): δ 197.7, 165.9, 164.6, 149.1, 148.4, 144.7, 139.4, 138.4, 135.8, 135.6, 134.8, 133.7, 132.2,
130.8, 130.2, 129.8, 129.4, 128.6, 128.1 (3C), 101.7, 66.4, 62.5, 55.9, 44.9, 38.8, 21.0; HRMS (MS-TOF):
[M+H]+ calcd. for C32H29IN3O5S: 694.0867, found: 694.0864. cis-6d: Colorless amorphous solid
(75 mg, 5%): mp 115–118 °C; IR ν 2933, 1657, 1596, 1579, 1421, 1323, 1140, 1085, 1012, 814, 754,
688 cm−1; 1H-NMR (400 MHz, DMSO) δ 8.47 (dd, 4.8 Hz, 1.9 Hz, 1H), 8.26 (d, 1.9 Hz, 1H), 8.10 (dd,
7.7 Hz, 1.2 Hz, 2H), 7.82 (dd, 7.8 Hz, 1.1 Hz, 1H), 7.78 (dd, 7.8 Hz, 1.3 Hz, 1H), 7.70 (tt, 7.7 Hz,
1.2 Hz, 1H), 7.62–7.57 (m, 4H), 7.52 (dt, 8.0 Hz, 1.9 Hz, 1H), 7.45 (td, 7.8 Hz, 1.1 Hz, 1H), 7.35 (dd,
8.0 Hz, 4.8 Hz, 1H), 7.20 (d, 8.0 Hz, 2H), 7.08 (td, 7.8 Hz, 1.3 Hz, 1H), 5.14 (s, 1H), 5.11 (d, 14.5 Hz,
1H), 5.04 (dd, 4.4 Hz, 3.9 Hz, 1H), 5.00 (d, 14.5 Hz, 1H), 4.85 (d, 15.4 Hz, 1H), 4.19 (dd, 18.9 Hz,
4.4 Hz, 1H), 3.99 (dd, 18.6 Hz, 3.9 Hz, 1H), 3.73 (d, 15.4 Hz, 1H), 2.27 (s, 3H); 13C-NMR (100 MHz,
DMSO): δ 196.9, 165.2, 164.0, 149.0, 148.5, 144.8, 139.4, 138.3, 136.1, 135.6, 134.0, 133.4, 131.1,
130.3, 129.7, 129.0, 128.8, 128.6, 128.3, 128.1, 123.4, 102.5, 66.0, 63.9, 55.9, 44.9, 40.8, 21.1; HRMS
(MS-TOF): [M+H]+ calcd. for C32H29IN3O5S: 694.0867, found: 694.0868.
3-(4-Fluorophenyl)-6-(2-oxo-2-phenylethyl)-4-(pyridin-3-ylmethyl)-1-(tosylmethyl)piperazine-2,5-dione
(trans-6e). Quantities: 4-fluorobenzaldehyde (220 µL, 2.0 mmol), 3-(aminomethyl)pyridine
(200 µL, 2.0 mmol), p-toluenesulfonylmethyl isocyanide (390 mg, 2.0 mmol), 3-benzoylacrylic acid
(350 mg, 2.0 mmol). The precipitate was filtered and washed with cold methanol to give pure trans-6e.
Colorless solid (494 mg, 42%): mp 174–175 °C; IR ν 2984, 1684, 1652, 1597, 1579, 1510, 1422, 1329,
1
1221, 1145, 1088, 812, 753, 687 cm−1; H-NMR (400 MHz, DMSO) δ 8.41 (dd, 4.9 Hz, 1.8 Hz, 1H),
8.17 (d, 1.8 Hz, 1H), 7.98 (d, 7.7 Hz, 2H), 7.69 (tt, 7.7 Hz, 1.1 Hz, 1H), 7.65 (d, 8.3 Hz, 2H), 7.57 (t,
7.7 Hz, 2H), 7.41 (dt, 8.0 Hz, 1.8 Hz, 1H), 7.28 (d, 8.0 Hz, 2H), 7.25 (dd, 8.0 Hz, 4.9 Hz, 1H), 7.12 (d,
7.3 Hz, 4H), 5.49 (d, 14.9 Hz, 1H), 5.24 (s, 1H), 4.93 (dd, 4.2 Hz, 3.5 Hz, 1H), 4.88 (d, 14.9 Hz, 1H),
4.82 (d, 15.3 Hz, 1H), 4.25 (dd, 18.9 Hz, 4.2 Hz, 1H), 3.89 (dd, 18.9 Hz, 3.5 Hz, 1H), 3.84 (d, 15.3 Hz,
1H), 2.34 (s, 3H); 13C-NMR (100 MHz, DMSO): δ 197.4, 165.6, 165.3, 161.9 (d, 1J(C,F) = 244.9 Hz),
4
149.0, 148.3, 144.7, 135.9, 135.5, 134.5, 133.6, 132.1 (d, J(C,F) = 3.0 Hz), 131.4, 130.2 (d,
3J(C,F) = 8.6 Hz), 129.7, 128.6, 128.0 (2C), 123.1, 115.2 (d, 2J(C,F) = 21.9 Hz), 62.7, 61.5, 55.4, 45.2,
38.0, 20.9; LCMS (ESI+): m/z 586.0 [M+H]+; Anal. calcd. for C32H28FN3O5S: C 65.63, H 4.82, N 7.18,
S 5.48; found: C 65.95, H 4.88, N 7.27, S 5.36.
1-Benzyl-3-(2-oxo-2-phenylethyl)-6-phenyl-4-(tosylmethyl)piperazine-2,5-dione (trans-6f). Quantities:
benzaldehyde (200 µL, 2.0 mmol), benzylamine (220 µL, 2.0 mmol), p-toluenesulfonylmethyl
isocyanide (390 mg, 2.0 mmol), 3-benzoylacrylic acid (350 mg, 2.0 mmol). The precipitate was
filtered and washed with cold methanol to give pure trans-6f. Colorless solid (628 mg, 55%): mp
201–203 °C; IR ν 2929, 1662, 1598, 1421, 1318, 1143, 1087, 814, 749, 691 cm−1; 1H-NMR (400 MHz,
DMSO) δ 8.01 (dd, 7.4 Hz, 1.5 Hz, 2H), 7.69 (tt, 7.4 Hz, 1.5 Hz, 1H), 7.62 (d, 8.3 Hz, 2H), 7.58 (t, 7.4 Hz,
2H), 7.41–7.31 (m, 3H), 7.27–7.22 (m, 5H), 7.05 (dd, 7.8 Hz, 1.4 Hz, 2H), 7.00 (dd, 6.6 Hz, 2.9 Hz,
2H), 5.48 (d, 14.9 Hz, 1H), 5.05 (d, 15.2 Hz, 1H), 5.04 (s, 1H), 4.92 (t, 3.7 Hz, 1H), 4.87 (d, 14.9 Hz,
1H), 4.20 (dd, 18.8 Hz, 3.7 Hz, 1H), 3.90 (dd, 18.8 Hz, 3.7 Hz, 1H), 3.57 (d, 15.2 Hz, 1H), 2.34
(s, 3H); 13C NMR (100 MHz, DMSO): δ 197.2, 165.5, 165.4, 144.7, 136.0, 135.5, 135.4, 134.5, 133.6,

Molecules 2012, 17
14695
129.7, 128.6, 128.5, 128.3, 128.2, 128.02, 127.97, 127.7 (2C), 127.2, 62.9, 61.5, 55.4, 46.9, 37.7, 21.0;
LCMS (ESI+): m/z 566.8 [M]+; Anal. calcd. for C33H30N2O5S: C 69.94, H 5.34, N 4.94, S 5.66; found:
C 70.10, H 5.35, N 5.07, S 5.60.
3-(2-Oxo-2-phenylethyl)-1-phenethyl-6-phenyl-4-(tosylmethyl)piperazine-2,5-dione
(trans-6g).
Quantities: benzaldehyde (200 µL, 2.0 mmol), phenethylamine (250 µL, 2.0 mmol),
p-toluenesulfonylmethyl isocyanide (390 mg, 2.0 mmol), 3-benzoylacrylic acid (350 mg, 2.0 mmol).
The precipitate was filtered, dissolved in dichloromethane and extracted with aqueous saturated solution
of sodium hydrogen carbonate to give pure trans-6g. Colorless solid (135 mg, 12%): mp 198–201 °C;
1
IR ν 2931, 1685, 1655, 1597, 1427, 1326, 1142, 1087, 813, 752, 689 cm−1; H-NMR (400 MHz,
DMSO) δ 8.02 (dd, 7.5 Hz, 1.3 Hz, 2H), 7.69 (tt, 7.5 Hz, 1.3 Hz, 1H), 7.65 (d, 8.2 Hz, 2H), 7.57 (t,
7.5 Hz, 2H), 7.39–7.34 (m, 3H), 7.31 (d, 8.2 Hz, 2H), 7.21 (t, 7.2 Hz, 2H), 7.17 (dd, 7.1 Hz, 2.2 Hz,
2H), 7.14 (tt, 7.2 Hz, 1.4 Hz, 1H), 7.00 (dd, 7.2 Hz, 1.4 Hz, 2H), 5.49 (d, 14.8 Hz, 1H), 5.36 (s, 1H),
4.88 (d, 14.8 Hz, 1H), 4.84 (dd, 4.4 Hz, 3.6 Hz, 1H), 4.15 (dd, 18.7 Hz, 4.4 Hz, 1H), 3.83 (dd, 18.7 Hz,
3.6 Hz, 1H), 3.71 (ddd, 13.3 Hz, 10.7 Hz, 5.3 Hz, 1H), 2.80 (ddd, 12.7 Hz, 10.5 Hz, 5.3 Hz, 1H), 2.65
(ddd, 13.3 Hz, 10.5 Hz, 5.6 Hz, 1H), 2.40 (ddd, 12.7 Hz, 10.7 Hz, 5.6 Hz, 1H), 2.36 (s, 3H); 13C-NMR
(100 MHz, DMSO): δ 197.1, 165.8, 164.9, 144.7, 138.5, 136.6, 135.9, 134.6, 133.6, 129.8, 128.6,
128.5, 128.33, 128.28, 128.2, 128.1, 128.0 (2C), 126.1, 63.2, 61.8, 55.4, 46.1, 38.1, 31.6, 21.0; Anal.
calcd. for C34H32N2O5S: C 70.32, H 5.55, N 4.82, S 5.52; found: C 70.44, H 5.68, N 4.93, S 5.52.
1-(4-Methoxybenzyl)-3-(2-oxo-2-phenylethyl)-6-phenyl-4-(tosylmethyl)piperazine-2,5-dione
(trans-
6h). Quantities: benzaldehyde (200 µL, 2.0 mmol), 4-methoxybenzylamine (260 µL, 2.0 mmol),
p-toluenesulfonylmethyl isocyanide (390 mg, 2.0 mmol), 3-benzoylacrylic acid (350 mg, 2.0 mmol).
The precipitate was filtered and washed with cold methanol to give pure trans-6h. Colorless solid
(460 mg, 39%): mp 241–243 °C; IR ν 2930, 1659, 1611, 1598, 1611, 1598, 1510, 1432, 1318, 1247,
1
1178, 1144, 1088, 824, 754, 690 cm-1; H-NMR (400 MHz, DMSO) δ 8.00 (dd, 7.6 Hz, 1.2 Hz, 2H),
7.69 (tt, 7.6 Hz, 1.2 Hz, 1H), 7.60 (d, 8.1 Hz, 2H), 7.58 (t, 7.6 Hz, 2H), 7.42–7.33 (m, 3H), 7.24 (d, 8.1 Hz,
2 H), 7.05 (dd, 7.7 Hz, 1.6 Hz, 2H), 6.92 (d, 8.7 Hz, 2H), 6.80 (d, 8.7 Hz, 2H), 5.47 (d, 14.9 Hz, 1H),
5.04 (d, 14.9 Hz, 1H), 4.98 (s, 1H), 4.90 (dd, 4.5 Hz, 3.7 Hz, 1H), 4.86 (d, 14.9 Hz, 1H), 4.18 (dd,
18.7 Hz, 4.5 Hz, 1H), 3.88 (dd, 18.7 Hz, 3.7 Hz, 1H), 3.72 (s, 3H), 3.44 (d, 14.9 Hz, 1H), 2.33 (s, 3H);
13C-NMR (100 MHz, DMSO): δ 197.1, 165.6, 165.3, 158.5, 144.6, 136.0, 135.6, 134.5, 133.5, 129.7,
129.3, 128.6, 128.5, 128.2, 128.02, 127.96, 127.7, 127.1, 113.6, 62.6, 61.5, 55.4, 54.9, 46.2, 37.6, 21.0;
LCMS (ESI+): m/z 596.4 [M]+; Anal. calcd. for C34H32N2O6S: C 68.44, H 5.41, N 4.69, S 5.37; found:
C 68.47, H 5.43, N 4.86, S 5.40.
3-(2-(4-Chlorophenyl)-2-oxoethyl)-6-phenyl-1-(pyridin-3-ylmethyl)-4-(tosylmethyl)piperazine-2,5-dione
(trans-6i). Quantities: benzaldehyde (200 µL, 2.0 mmol), 3-(aminomethyl)pyridine (200 µL,
2.0 mmol), p-toluenesulfonylmethyl isocyanide (390 mg, 2.0 mmol), 3-(4-chlorobenzoyl)acrylic acid
(420 mg, 2.0 mmol). The precipitate was filtered, dissolved in ethyl acetate and crystallization initiated
by adding n-hexane to give pure trans-6i. Colorless solid (449 mg, 37%): mp 233–234 °C; IR ν 2940,
1
1686, 1651, 1591, 1576, 1435, 1325, 1142, 815, 754, 699 cm−1; H-NMR (400 MHz, DMSO) δ 8.46
(dd, 4.7 Hz, 1.8 Hz, 1H), 8.17 (d, 1.8 Hz, 1H), 7.99 (ddd, 8.6 Hz, 2.4 Hz, 1.8 Hz, 2H), 7.64 (ddd, 8.8
Hz, 2.4 Hz, 1.8 Hz, 2H), 7.62 (d, 8.5 Hz, 2H), 7.41 (dt, 7.9 Hz, 1.8 Hz, 1H), 7.37–7.29 (m, 3H), 7.26
(d, 8.5 Hz, 2H), 7.26–7.23 (m, 1H), 7.07 (dd, 7.8 Hz, 1.1 Hz, 2H), 5.46 (d, 14.9 Hz, 1H), 5.18 (s, 1H),

Molecules 2012, 17
14696
4.93 (dd, 4.4 Hz, 3.6 Hz, 1H), 4.88 (d , 14.9 Hz, 1H), 4.86 (d, 15.3 Hz, 1H), 4.21 (dd, 18.9 Hz, 4.4 Hz,
1H), 3.88 (dd, 18.9 Hz, 3.6 Hz, 1H), 3.79 (d, 15.3 Hz, 1H), 2.34 (s, 3H); 13C-NMR (100 MHz,
DMSO): δ 196.4, 165.6, 165.4, 149.0, 148.3, 144.7, 128.7, 138.5, 135.6, 135.4, 134.6, 134.5, 131.4,
129.9, 129.7, 128.5, 128.3, 127.98, 127.92, 123.2, 63.4, 61.7, 55.4, 45.3, 38.0, 21.0; LCMS (ESI+): m/z
602.0 [M]+; HRMS (MS-TOF): [M+H]+ calcd. for C32H29ClN3O5S: 602.1511, found: 602.1515.
3-(2-(4-Methoxyphenyl)-2-oxoethyl)-6-phenyl-1-(pyridin-3-ylmethyl)-4-(tosylmethyl)piperazine-2,5-
dione (trans-6j). Quantities: benzaldehyde (200 µL, 2.0 mmol), 3-(aminomethyl)pyridine (200 µL,
2.0 mmol), p-toluenesulfonylmethyl isocyanide (390 mg, 2.0 mmol), 3-(4-methoxybenzoyl)acrylic
acid (410 mg, 2.0 mmol). The precipitate was filtered and washed with cold methanol to give pure
trans-6j. Colorless solid (434 mg, 36%): mp 250–252 °C; IR ν 2938, 1650, 1601, 1576, 1510, 1437,
1
1327, 1261, 1171, 1142, 1089, 825, 754, 699 cm−1; H-NMR (400 MHz, DMSO) δ 8.41 (dd, 4.8 Hz,
1.8 Hz, 1H), 8.15 (d, 1.8 Hz, 1H), 7.97 (d, 9.0 Hz, 2H), 7.64 (d, 8.2 Hz, 2H), 7.39 (dt, 7.9 Hz, 1. 8 Hz,
1H), 7.36-7.30 (m, 3H), 7.24 (dd, 7.9 Hz, 4.8 Hz, 1H), 7.28 (d, 8.2 Hz, 2H), 7.08 (d, 9.0 Hz, 2H), 7.06
(dd, 8.3 Hz, 1.4 Hz, 2H), 5.48 (d, 14.8 Hz, 1H), 5.17 (s, 1H), 4.89 (dd, 4.3 Hz, 3.2 Hz, 1H), 4.85 (d,
15.4 Hz, 1H), 4.84 (d, 14.8 Hz, 1H), 4.18 (18.7 Hz, 4.3 Hz, 1H), 3.87 (s, 3H), 3.81 (18.7 Hz, 3.2 Hz,
1H), 3.78 (d, 15.4 Hz, 1H), 2.34 (s, 3H); 13C NMR (100 M Hz, DMSO): δ 195.6, 165.8, 165.6, 163.5,
149.0, 148.3, 144.7, 136.0, 135.4, 134.6, 131.4, 130.4, 129.8, 128.9, 128.4, 128.3, 128.04, 128.00,
123.1, 113.8, 63.4, 61.6, 55.52, 55.50, 45.2, 37.5, 21.0; LCMS (ESI+): m/z 598.2 [M+H]+; HRMS
(MS-TOF): [M+H]+ calcd. for C33H32N3O6S: 598.2006, found: 598.2009.
3.4. Bioactivity Analyses
3.4.1. Isolation and Activation of T Cells
T cells were isolated from the peripheral blood of human healthy donors by Biocoll density gradient
centrifugation (Biochrom, Berlin, Germany) and subsequent positive isolation using magnetic cell
sorting techniques (Miltenyi Biotec, Bergisch-Gladbach, Germany). For activation, T cells were
cocultured for 5 days with allogeneic dendritic cells (DCs) in culture medium supplemented with 10%
heat-inactivated, pooled human serum and 100 U/mL penicillin-streptomycin (Invitrogen, Darmstadt,
Germany). DCs were generated within 3 days from isolated monocytes according to a previously
described protocol [24] and matured by addition of 25 ng/mL tumor necrosis factor-α, 5 ng/mL IL-1β,
10 ng/mL IL-6 (all R&D Systems, Wiesbaden, Germany), and 1 µg/mL prostaglandin E2
(Sigma-Aldrich, Munich, Germany) for 24 h to the culture medium. Newly synthesized compounds
were added at day 4 to the activation culture for the last 24 h.
3.4.2. Analysis of T Cell Proliferation
T cell proliferation was quantified by measuring [3H]thymidine (GE healthcare, Munich, Germany)
incorporation. Synthesized compounds and 17-DMAG (Invivogen, Toulouse, France) were resolved in
DMSO (Sigma-Aldrich, Munich, Germany). Activated T cells were incubated on day 4 for the last
24 h with the indicated concentrations of synthesized compounds, 17-DMAG or DMSO control and
pulsed with 5µCi/mL [3H]thymidine for the last 18 h. Incorporation of [3H]thymidine was quantified
using a beta-counter (Perkin-Elmer, Rodgau, Germany).

Molecules 2012, 17
14697
3.4.3. Analysis of Apoptosis
Apoptotic cell death was determined using AnnexinV-Cy5/7-AAD staining. Briefly, resting T cells
were treated for 24 h with the indicated concentrations of the synthesized compounds, 17-DMAG or
DMSO ctrl washed with phosphate-buffered saline and resuspended in binding buffer in a
concentration of 1 × 106 cells/mL. Annexin V-Cy5 (BD biosciences, Heidelberg, Germany) and
7-AAD (ebioscience, Frankfurt, Germany) were added and cells were incubated for 15 min at room
temperature in the dark. Apoptosis of cells was measured and quantified using flow cytometry.
Percentages of specific apoptosis (sA) were calculated as follows:
AE  AC
sA(%) 100
100 AC
where AE equals % of apoptotic cells in the experimental group, and AC equals % of apoptotic cells in
the control group.
3.4.4. Statistical Analyses
Statistical analyses were performed using Microsoft EXCEL software (Munich, Germany).
Differences between mean values were assessed using two-tailed paired Student’s t-test. Statistical
significance was set at p < 0.05.
3.5. X-ray Crystallography
The single-crystals of compound 6b were obtained by slow diffusion of petroleum ether to a
compound solution in dichloromethane. Intensity data were collected at a Temperature of 173 K on a
Bruker APEX-CCD (D8 three-circle goniometer) (Bruker AXS) diffractometer with graphite
monochromated Mo-Kα radiation (λ = 0.71073 Å) using the ω scan mode with 1.74° < θ < 25.00°;
20749 reflections were measured with 4764 unique reflections. 3326 reflections with I > 2σ(I) were
used in the Fourier techniques. The final refinement was converged to R1 = 0.0919 [I > 2(I)],
2
wR2(Fo ) = 0.2499 (all data). Crystal data: empirical formula C32H29N3O5S, crystal dimension
0.24 × 0.06 × 0.05 mm, monoclinic, space group P21/n, a = 5.7974(8) Å, b = 23.460(3) Å,
c = 20.101(3) Å, β = 96.219(3) °, V = 2717.8(6) Å3, Mr = 567.64, Z = 4, Dc = 1.387 Mg/m3,
μ(Mo Kα) = 0.168 mm−1, F(000) = 1192, S = 1.096. Crystallographic data (excluding structure factors)
have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication
No. CCDC-910534. Copies of the data can be obtained free of charge on application to Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; [fax: (+44)-1223-336-033;
email: deposit@ccdc.cam.ac.uk]. Details of the crystal structure are given in the crystallographic figure
and data tables (Supplementary Figure 1, Tables 2 and 3).
4. Conclusions
In summary, we were able to synthesize a library of differently substituted 2,5-diketopiperazines via
a coupled Ugi/Aza-Michael reaction by using β-acyl substituted acrylic acids. The stereochemistry of
the highly substituted ring system was confirmed by X-ray crystallography and NMR spectroscopy.

Molecules 2012, 17
14698
The small library of 2,5-diketopiperazines was analysed with respect to their anti-proliferative
properties. Proliferation analyses using activated human T cells demonstrated a significant
anti-proliferative effect of the 2,5-diketopiperazine trans-6a, trans-6c, trans-6d in noncytotoxic
concentrations. Detailed experiments analysing apoptosis and cell cycle in activated T cells to explore
the underlying mechanism of the observed anti-proliferative effect are in progress.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/12/14685/s1.
Acknowledgments
Financial support by the DFG (Clinical Research Unit 216) is gratefully acknowledged.
References
1. Domling, A.; Ugi, I. Multicomponent reactions with isocyanides. Angew. Chem. Int. Ed. 2000, 39,
3168–3210.
2. Ugi, I.; Steinbrueckner, C. Aminocarboxylic acid amides and derivatives. DE 1103337, 30 March 1961.
3. Hulme, C.; Gore, V. Multi-component reactions: Emerging chemistry in drug discovery from
xylocain to crixivan. Curr. Med. Chem. 2003, 10, 51–80.
4. Ugi, I.; Steinbrückner, C. Über ein neues Kondensations-Prinzip. Angew. Chem. 1960, 72, 267–268.
5. Borthwick, A.D. 2,5-Diketopiperazines: Synthesis, Reactions, Medicinal Chemistry, and
Bioactive Natural Products. Chem. Rev. (Washington, DC, U. S.) 2012, 112, 3641–3716.
6. Scharf, D.H.; Chankhamjon, P.; Scherlach, K.; Heinekamp, T.; Roth, M.; Brakhage, A.A.;
Hertweck, C. Epidithiol Formation by an Unprecedented Twin Carbon-Sulfur Lyase in the
Gliotoxin Pathway. Angew. Chem. Int. Ed. 2012, 51, 9943.
7. Borthwick, A.D.; Davies, D.E.; Exall, A.M.; Hatley, R.J.D.; Hughes, J.A.; Irving, W.R.;
Livermore, D.G.; Sollis, S.L.; Nerozzi, F.; Valko, K.L.; et al. 2,5-Diketopiperazines as Potent,
Selective, and Orally Bioavailable Oxytocin Antagonists. 3. Synthesis, Pharmacokinetics, and in
Vivo Potency. J. Med. Chem. 2006, 49, 4159–4170.
8. Zeng, Y.; Li, Q.; Hanzlik, R.P.; Aube, J. Synthesis of a small library of diketopiperazines as
potential inhibitors of calpain. Bioorg. Med. Chem. Lett. 2005, 15, 3034–3038.
9. Wang, D.-X.; Liang, M.-T.; Tian, G.-J.; Lin, H.; Liu, H.-Q. A facile pathway to synthesize
diketopiperazine derivatives. Tetrahedron Lett. 2002, 43, 865–867.
10. Endo, A.; Yanagisawa, A.; Abe, M.; Tohma, S.; Kan, T.; Fukuyama, T. Total Synthesis of
Ecteinascidin 743. J. Am. Chem. Soc. 2002, 124, 6552–6554.
11. Dinsmore, C.J.; Beshore, D.C. Recent advances in the synthesis of diketopiperazines. Tetrahedron
2002, 58, 3297–3312.
12. Santra, S.; Andreana, P.R. A One-Pot, Microwave-Influenced Synthesis of Diverse Small
Molecules by Multicomponent Reaction Cascades. Org. Lett. 2007, 9, 5035–5038.

Molecules 2012, 17
14699
13. Santra, S.; Andreana, P.R. A Rapid, One-Pot, Microwave-Influenced Synthesis of Spiro-2,5-
diketopiperazines via a Cascade Ugi/6-Exo-Trig Aza-Michael Reaction. J. Org. Chem. 2011, 76,
2261–2264.
14. Portal, C.; Launay, D.; Merritt, A.; Bradley, M. High Throughput Physical Organic Chemistry:
Analytical Constructs for Monomer Reactivity Profiling. J. Comb. Chem. 2005, 7, 554–560.
15. Mehta, V.P.; Modha, S.G.; Ruijter, E.; Van Hecke, K.; Van Meervelt, L.; Pannecouque, C.;
Balzarini, J.; Orru, R.V.A.; van der Eycken, E. A Microwave-Assisted Diastereoselective
Multicomponent Reaction To Access Dibenzo[c,e]azepinones: Synthesis and Biological
Evaluation. J. Org. Chem. 2011, 76, 2828–2839.
16. Leeson, P.D.; Oprea, T.I. Drug-like physicochemical properties. RSC Drug Discov. Ser. 2012, 13,
35–59.
17. Mofakham, H.; Hezarkhani, Z.; Shaabani, A. Cellulose-SO3H as a biodegradable solid acid
catalyzed one-pot three-component Ugi reaction: Synthesis of α-amino amide, 3,4-
dihydroquinoxalin-2-amine, 4H-benzo[b][1,4]thiazin-2-amine and 1,6-dihydropyrazine-2,3-
dicarbonitrile derivatives. J. Mol. Catal. A Chem. 2012, 360, 26–34.
18. Davies, S.G.; Christopher Garner, A.; Ouzman, J.V.A.; Roberts, P.M.; Smith, A.D.; Snow, E.J.;
Thomson, J.E.; Tamayo, J.A.; Vickers, R.J. Diastereoselective synthesis of quaternary α-amino
acids from diketopiperazine templates. Org. Biomol. Chem. 2007, 5, 2138–2147.
19. Gomez-Monterrey, I.; Campiglia, P.; Carotenuto, A.; Califano, D.; Pisano, C.; Vesci, L.; Lama, T.;
Bertamino, A.; Sala, M.; Di Bosco, A.M.; et al. Design, Synthesis, and Cytotoxic Evaluation of a
New Series of 3-Substituted Spiro[(dihydropyrazine-2,5-dione)-6,3'-(2',3'-dihydrothieno[2,3-
b]naphthalene-4',9'-dione)] Derivatives. J. Med. Chem. 2007, 50, 1787–1798.
20. Gomez-Monterrey, I.; Campiglia, P.; Carotenuto, A.; Stiuso, P.; Bertamino, A.; Sala, M.;
Aquino, C.; Grieco, P.; Morello, S.; Pinto, A.; et al. Spiro[(dihydropyrazin-2,5-dione)-6,3'-(2',3'-
dihydrothieno[2,3-b]naphtho-4',9'-dione)]-Based Cytotoxic Agents: Structure-Activity Relationship
Studies on the Substituent at N4-Position of the Diketopiperazine Domain. J. Med. Chem. 2008,
51, 2924–2932.
21. Sayegh, M.H.; Turka, L.A. The role of T-cell costimulatory activation pathways in transplant
rejection. N. Engl. J. Med. 1998, 338, 1813–1821.
22. Skapenko, A.; Leipe, J.; Lipsky Peter, E.; Schulze-Koops, H. The role of the T cell in autoimmune
inflammation. Arthrit. Res. Ther. 2005, 7, S4–S14.
23. Stuehler, C.; Mielke, S.; Chatterjee, M.; Duell, J.; Lurati, S.; Rueckert, F.; Einsele, H.; Bargou, R.C.;
Topp, M.S. Selective depletion of alloreactive T cells by targeted therapy of heat shock protein
90: a novel strategy for control of graft-versus-host disease. Blood 2009, 114, 2829–2836.
24. Dauer, M.; Obermaier, B.; Herten, J.; Haerle, C.; Pohl, K.; Rothenfusser, S.; Schnurr, M.; Endres, S.;
Eigler, A. Mature dendritic cells derived from human monocytes within 48 hours: A novel strategy
for dendritic cell differentiation from blood precursors. J. Immunol. 2003, 170, 4069–4076.
Sample Availability: Samples of the synthesized compounds are available from the authors.
© 2012 by the authors; licensee MDPI, 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/).