Synthesis and cytotoxicity of the proposed structure of piperazirum, its stereoisomers and analogues

Article abstract of DOI:10.1002/ejoc.201301435

Piperazirum, a new biologically active alkaloid isolated from Arum palaestinum Bioss, is described, starting from commercially available α-amino acids. Two synthetic strategies were developed for the synthesis of the enantiomeric pair of compounds corresponding to the proposed structure of piperazirum. The first strategy was used in the synthesis of stereoisomers, and the second one was used in generating the analogues. The compounds synthesized were screened against different cell lines to evaluate their cytotoxicity profiles. Piperazirum, its stereoisomers, and its analogues were synthesized and tested for their antiproliferative activity. Copyright

Full text of DOI:10.1002/ejoc.201301435

FULL PAPER
DOI: 10.1002/ejoc.201301435
Synthesis and Cytotoxicity of the Proposed Structure of Piperazirum, Its
Stereoisomers and Analogues
Gangarajula Sudhakar,*[a] Shruthi Bayya,[a] Karla Janardhan Reddy,[a]
Balasubramanian Sridhar,[b] Komal Sharma,[c] and Surendar Reddy Bathula*[c]
Keywords: Natural products / Total synthesis / Alkaloids / Nitrogen heterocycles / Medicinal chemistry / Cytotoxicity
Piperazirum, a new biologically active alkaloid isolated from
Arum palaestinum Bioss, is described, starting from commer-
cially available α-amino acids. Two synthetic strategies were
developed for the synthesis of the enantiomeric pair of com-
pounds corresponding to the proposed structure of piperaz-
irum. The first strategy was used in the synthesis of stereoiso-
mers, and the second one was used in generating the ana-
logues. The compounds synthesized were screened against
different cell lines to evaluate their cytotoxicity profiles.
been published.[21] The first one describes a synthetic route
to 2-oxopiperazine derivatives targeting the natural product
piperazirum,[21a] and the second one, which appeared very
recently, describes the synthesis of the reported structure
of piperazirum.[21b] However, the correct structure and the
absolute configuration of this compound are not yet
known.
Introduction
The piperazine and piperazinone motifs are considered
to be privileged structures in drug discovery.[1] Such motifs
are found in the structures of several natural products that
show a wide range of biological activities, such as pseudo-
theonamides A1, A2, and B3,[2] dragmacidin[3] and dragmac-
idins A–C,[4] dihydrohamacanthin A,[5] ecteinascidin 743,[6]
TAN1251A,[7] agelastatin A,[8] marcfortine B,[9] phakel-
lins,[10] and guadinomine C2.[11] This ring is also present in
compounds with antifungal,[12] antidepressant,[13] antimig-
raine,[14] antithrombotic,[15] antihistaminic,[16] antiaggregat-
ing,[17] or nootropic[18] activities. In 2007, piperazirum, a
new biologically active alkaloid, was isolated from the n-
butanol-soluble fraction of the leaf extract o Arum palaesti-
num Bioss.[19] It was found to show significant inhibition of
all the cultured human cell lines examined using the SRB
(sulforhodamine B) method.[20] These cell lines included
A549 (non-small cell lung, ED50 = 4.26Ϯ0.2 μm), SK-OV-
3 (ovary, ED50 = 1.38Ϯ0.1 μm), SK-MEL-2 (melanoma,
The unknown absolute stereochemistry, the highly sub-
stituted chiral 2-oxopiperazine structure, and the promising
cytotoxicity of piperazirum attracted us to develop a strat-
egy for its synthesis. We aimed to develop a strategy for the
synthesis of this molecule that would not only provide
enough material for further biological evaluation of piper-
azirum, but that would also enable access to stereoisomers
and analogues that could be used to study a structure–ac-
tivity relationship (SAR) and so find more potent mole-
cules. In this paper, we describe the synthesis of the pro-
posed structure of piperazirum, and also its stereoisomers
and analogues. We also give the results of testing their in
vitro antiproliferative activity against seven different cell
lines, including A549 (human lung adenocarcinoma epithe-
lial cell line), SK-OV-3 (ovarian carcinoma cell line), DU-
145 (human prostate cancer cell line), MDA-MB-231 and
MCF-7 (human breast cancer cell lines), HEK-293 (human
embryonic kidney cells), and NIH/3T3 (mouse embryo fi-
broblast cell line).
ED50 = 0.51Ϯ0.1 μm), and HCT-15 (colon, ED50
=
2.47Ϯ0.3 μm). The structure and relative stereochemistry
of piperazirum were deduced on the basis of 1D and 2D
NMR spectroscopic data. To the best of our knowledge,
only two reports of the synthesis of this compound have
[a] Division of CPC (Organic Chemistry-II), CSIR – Indian
Institute of Chemical Technology,
Tarnaka, Hyderabad 500007, India
E-mail: gsudhakar@iict.res.in
Results and Discussion
http://www.iictindia.org/
[b] Centre for X-ray Crystallography, CSIR – Indian Institute of
Chemical Technology,
Our retrosynthetic analysis of 1 is shown in Figure 1. To
get the syn stereochemistry at C-3, C-5, and C-6, we chose
acyclic amide 2, in which the amine, upon deprotection of
the Boc (tert-butoxycarbonyl) group, could displace the
leaving group (X) in an SN2 fashion to give the target mo-
lecule under basic conditions. Acyclic compound 2 could
Tarnaka, Hyderabad 500007, India
[c] Pharmaceutics Division, CSIR – Central Drug Research
Institute,
Lucknow 226031, U.P., India
E-mail: bsreddy@cdri.res.in
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301435.
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G. Sudhakar, S. R. Bathula et al.
FULL PAPER
easily be derived from the coupling of 1,2-diamine deriva-
tive 3 and α-hydroxy acid 4. These two fragments could be
made from the same starting material, l-leucine.
Figure 1. Retrosynthetic analysis of the proposed structure of pip-
erazirum (1).
The synthesis began with a reduction of 5[22] using
DIBAL-H (diisobutylaluminium hydride), to convert the
ester into an aldehyde (Scheme 1). The aldehyde was treated
with isopropylmagnesium bromide to give a separable mix-
ture of diastereomers 6 and 6a (3.5:1 ratio) in 85% com-
bined yield. Mesylation of 6, the major isomer, with MsCl
and Et3N in CH2Cl2 followed by displacement of the mesyl
group with sodium azide gave the corresponding amino az-
ide (i.e., 7) in 83% yield. Hydrogenation of 7 under balloon
pressure in the presence of Pd/C in a mixture of EtOAc/
MeOH (2:1) gave amine 3 in quantitative yield. Primary
amine 3 was coupled to α-hydroxy acid (–)-4, which had
been derived from l-leucine by the diazotization method,[23]
in the presence of EDCI [1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide]/HOBt to give amide 8. The free hy-
droxy group in 8 was mesylated to give 9. Finally, removal
Scheme 1. Synthesis of 1 and 1a.
of the Boc protecting group with trifluoroacetic acid (TFA), the presence of DIPEA in MeOH resulted the formation of
followed by heating at reflux in the presence of DIPEA (di- the expected cyclic product (i.e., 1a).
isopropylethylamine) gave the desired compound (i.e., 1).
Other stereoisomers were planned from coupling of 3
We could not compare the NMR spectroscopic data of syn- and 3a, independently, with α-hydroxy acid (+)-4,[25] de-
thetic 1 with the data reported for the natural product due rived from d-leucine, to give 8b and 8c, respectively
to the insolubility of the synthetic sample in D2O, the sol- (Scheme 2). Treatment of 8b and 8c with mesyl chloride un-
vent for which the NMR spectroscopic data of the natural der basic conditions gave 9b and 9c, respectively. Upon Boc
product was reported.[24]
group deprotection and subsequent heating at reflux in
With this solubility discrepancy in mind, we speculated MeOH in the presence of DIPEA, 9b and 9c provided 1b
that the substituents at C-3, C-5, and C-6 on the piperazine and 1c, respectively. To our surprise, none of these isomers
ring may not have the syn relationship proposed by the iso- (1a–1c) was soluble in D2O, so we could not compare the
lation group.[19] We thought that stereoisomers in which the spectroscopic data with that of the natural product, which
substituents had an anti relationship may differ in their sol- hindered the confirmation of the structure as well as finding
ubilities, and so the synthesis of these compounds may help the absolute configuration of the natural product.[24b]
to find the absolute configuration of piperazirum.
As piperazirum was reported to have significant cytotox-
Compound 6a was mesylated, and subsequent displace- icity against cancer cell lines,[19] we expected that the syn-
ment of the mesyl group with sodium azide gave the corre- thesis of analogues of 1 could result in our finding potent
sponding amino azide (i.e., 7a; Scheme 1). Azide 7a was molecules based on a structure–activity relationship. Work-
treated with hydrogen at atmospheric pressure in the pres- ing towards this end, we envisioned another strategy for the
ence of Pd/C in a mixture of EtOAc/MeOH (2:1) to give synthesis of the enantiomer of 1 (ent-1), in order to access
3a. Under standard peptide-coupling conditions, 3a was analogues of 1 or its enantiomer by a shorter and more
coupled with acid (–)-4 to give 8a. The hydroxy group in 8a efficient route. This synthesis started with the reduction of
was mesylated with MsCl/Et3N, and subsequent removal of 10[26] using DIBAL-H, to convert the methyl ester into the
the Boc protecting group followed by heating at reflux in aldehyde. This aldehyde was then treated with isobutylmag-
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Synthesis and Cytotoxicity of Piperazirum
with hydrogen at atmospheric pressure in the presence of
Pd/C in MeOH. The Cbz group was removed, and the
imine formed intramolecularly was saturated to give the an-
ticipated ent-1 in excellent yield and with excellent dia-
stereoselectivity. The spectroscopic data of 1 and ent-1 are
identical in all respects.[21b] The specific rotations of 1 and
ent-1 have the same magnitude but opposite signs, which
indicates, as expected, that the two compounds have an en-
antiomeric relationship. The syn configuration of 1 and ent-
1 was further confirmed by the X-ray analysis of 15,[28] de-
rived from (–)-1, which made it clear that 1 or ent-1 is the
proposed structure of piperazirum. Comparison of the op-
tical rotation of all of the synthesized stereoisomers with
the natural product showed that only 1 {[α]2D6 = +52.5 (c =
0.33, MeOH)} had an optical rotation that was very close
to the one reported for natural product {[α]D = +50.0 (c =
0.024, MeOH)},[19] which indicates that 1 may be the cor-
rect structure for piperazirum. However, comparing the
NMR spectroscopic data was difficult because of the uncer-
tainty over the solvent (D2O or any other) used to record
the NMR spectra of the natural product.[24b]
After the successful completion of the short and efficient
synthetic route for rapid access to ent-1, we focussed our
attention on the synthesis of some higher analogues using
the same strategy. Accordingly, the free amine derived from
11 was coupled with 12a–12e [Cbz-l-CH(R)-OH, a: R = H;
b: R = CH3;[29] c: R = iPr; d: R = sec-Bu;[30] e: R = CH2Ph]
to give 16a–16e. Similarly, the free amine derived from 6
and 6a was coupled with 12a–12e and 12 [Cbz-l-CH(R)-
OH, R = iBu] to give 17a–17e and 17f, respectively
(Scheme 4). DMP oxidation of 16a–16e and 17a–17f gave
Scheme 2. Synthesis of 1b and 1c.
nesium bromide to give 11 (dr 7:1, based on 1H NMR spec-
troscopy; Scheme 3). Deprotection of the Boc protecting
group provided the free amine, which was coupled with 12
(Cbz-l-Leu-OH; Cbz = benzyloxycarbonyl)[27] in the pres-
ence of EDCI/HOBt to give dipeptide 13. Oxidation of the
secondary hydroxy group into a ketone using Dess–Martin
periodinane (DMP) produced 14. Finally, 14 was treated
Scheme 3. Synthesis of (–)-1 and 15; DMAP = 4-(dimethylamino)-
pyridine.
Scheme 4. Synthesis of 20a–20e and 21a–21f.
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Table 1. In-vitro anticancer activity (IC50 [μm]) of the proposed structure of piperazirum (+)-1, its enantiomer (–)-1, its diastereomers 1a–
1c, and its analogues (20a–20e, 21a–21f).
A549
SK-OV-3
DU-145
MDA-MB-231 MCF-7
HEK-293
NIH/3T3
(+)-1
(–)-1
1a
25Ϯ0.37
21Ϯ0.15
77Ϯ0.39
62Ϯ0.55
66Ϯ0.2
37Ϯ0.86
16Ϯ0.46
71Ϯ0.77
51Ϯ0.7
7 Ϯ0.33
86Ϯ0.78
70Ϯ0.97
44Ϯ0.98
82Ϯ0.52
32Ϯ0.69
43Ϯ0.61
19Ϯ0.37
17Ϯ0.92
43Ϯ0.91
54Ϯ0.76
42Ϯ0.19
18Ϯ0.51
9.3Ϯ0.39
28Ϯ0.62
59Ϯ0.47
4.4Ϯ0.13
57Ϯ0.69
46Ϯ0.58
52Ϯ0.57
90Ϯ0.58
17Ϯ0.86
41Ϯ0.41
45Ϯ0.24
36Ϯ0.21
81Ϯ0.43
73Ϯ0.17
92Ϯ0.10
61Ϯ0.20
22Ϯ0.26
80Ϯ0.16
77Ϯ0.32
10.5Ϯ0.2
93Ϯ0.29
89Ϯ0.12
72Ϯ0.35
81Ϯ0.59
51Ϯ0.63
61Ϯ0.61
50Ϯ0.23
40Ϯ0.27
78Ϯ0.19
77Ϯ0.07
87Ϯ0.19
63Ϯ0.24
19Ϯ0.24
82Ϯ0.16
67Ϯ0.49
19.2Ϯ0.1
91Ϯ0.45
90Ϯ0.52
71Ϯ0.25
89Ϯ0.54
52Ϯ0.63
69Ϯ0.57
40Ϯ0.22
26Ϯ0.22
86Ϯ0.13
69Ϯ0.31
90Ϯ0.12
67Ϯ0.24
28Ϯ0.54
89Ϯ0.12
89Ϯ0.42
14Ϯ0.3
96Ϯ0.21
99Ϯ0.32
78Ϯ0.45
95Ϯ0.50
55Ϯ0.65
62Ϯ0.87
59Ϯ0.20
53Ϯ0.26
95Ϯ0.10
69Ϯ0.46
98Ϯ0.17
63Ϯ0.22
48Ϯ0.14
87Ϯ0.19
89Ϯ0.02
34Ϯ0.23
92Ϯ0.76
87Ϯ0.37
76Ϯ0.40
95Ϯ0.50
59Ϯ0.05
60Ϯ0.17
61Ϯ0.01
63Ϯ0.16
89Ϯ0.16
78Ϯ0.66
89Ϯ0.16
61Ϯ0.21
52Ϯ0.12
83Ϯ0.29
81Ϯ0.12
37Ϯ0.28
88Ϯ0.36
83Ϯ0.34
71Ϯ0.20
81Ϯ0.59
52Ϯ0.15
65Ϯ0.11
1b
1c
20a
20b
20c
20d
20e
21a
21b
21c
21d
21e
21f
the corresponding ketones (i.e., 18a–18e and 19a–19f, of the stereoisomers 1a–1c showed significant cytotoxicity
respectively). Removal of the Cbz protecting group from against any of the cancer cell lines, except for 1a (43 μm)
18a–18e and 19a–19f and saturation of the subsequently and 1c (42 μm), which have some antiproliferative activity
formed imine under hydrogenation conditions gave substi- specifically against the SK-OV-3 cancer cell line.
tuted chiral 2-oxopiperazines 20a–20e and 21a–21f, respec-
tively.
The proposed structure of piperazirum (+)-1/(–)-1, its
diastereoisomers 1a–1c, and its analogues 20a–20e, and
21a–21f were tested for their antiproliferative activity, and
the results are summarized in Table 1.
Conclusions
In conclusion the enantiomeric pair corresponding to the
proposed structure of piperazirum was synthesized using
two different synthetic strategies. Stereoisomers and ana-
logues were synthesized following these strategies, and all
the compounds were evaluated for in vitro anticancer ac-
tivity against different cancer cell lines. X-ray analysis of 15
supports that 1 or ent-1 is the proposed structure of pipera-
zirum. The enantiomer of the proposed structure of pipera-
zirum and its analogues showed significant cytotoxicity
against ovarian cancer cell lines. Interestingly, 20e showed
selective anticancer activity against all the cancer cell lines
tested.
Biological Studies: Anti-Proliferative Activity
The proposed structure of piperazirum (+)-1/(–)-1, its
diastereoisomers 1a–1c, and its analogues 20a–20e, and
21a–21f were evaluated for in vitro anticancer activity using
an MTT assay across different concentrations (2.5, 5, 10,
20, 50, and 100 μm) in triplicate. The growth-inhibitory ef-
fects were studied in five human cancer cell lines, i.e., A549,
SK-OV-3, DU-145, MDA-MB-231, and MCF-7, and two
non-cancer cell lines, i.e., HEK-293 and NIH/3T3. Com-
pound 1 has IC50 values of 25, 19, 45, 50, 40, 59 and 61 μm
against A549, SK-OV-3, DU-145, MDA-MB-231, MCF-7,
HEK-293, and NIH/3T3, respectively (Table 1). Compound
(–)-1 also showed similar in vitro cytotoxic activity (Table 1)
against all the cell lines tested, and this enantiomer showed
the highest activity against SK-OV-3 cells, with an IC50
value of 17 μm. It is interesting to note that derivatives 20a–
Experimental Section
General Procedures: Anhydrous solvents were dried and distilled by
standard methods before use. Commercially sourced reagents were
used without further purification unless otherwise specified. All re-
actions were performed under an atmosphere of nitrogen or argon
in oven-dried glassware with magnetic stirring. Column chromatog-
raphy was carried out using silica gel (60–120, 100–200, or 230–
20e significantly inhibited the growth of ovarian cancer cell 400 mesh) and the column was eluted with ethyl acetate/petroleum
ether or with ethyl acetate. Analytical thin layer chromatography
(TLC) was performed on precoated silica gel-60 F254 (0.5 mm)
glass plates. Visualization of the spots on TLC plates was achieved
either using UV light or by staining the plates with ninhydrin/meth-
anol, methanolic anisaldehyde/sulfuric acid/acetic acid, or meth-
anol/phosphomolybdic acid/sulfuric acid solutions followed by
line (SK-OV-3) with IC50 values of 18 μm (20a), 9 μm (20b),
28 μm (20c), 59 μm (20d), and 4.4 μm (20e). Compound 20e
showed significant cytotoxicity against all the cancer cell
lines tested, with IC50 values of 7, 4.4, 10.5, 19.2, and 14 μm
against A549, SK-OV-3, DU-145, MDA-MB-231, and
MCF-7, respectively. Moreover, compound 20e showed se-
lective cytotoxicity towards cancer cells. Similarly, of ana-
logues 21a–21f, 21e (17 μm) has a better activity profile
specifically against SK-OV-3 cells than 21a (57 μm), 21b
1
charring on a hot plate. H and 13C NMR spectra were recorded
in CDCl3 with 500, 400, and 300 MHz spectrometers at ambient
temperature. Chemical shifts are reported on the δ scale, and were
calibrated using internal CHCl3 (δ = 7.26 ppm) or tetramethylsilane
(46 μm), 21c (52 μm), 21d (90 μm), and 21f (41 μm). None (δ = 0.0 ppm) for 1H NMR spectra, and CHCl3 (δ = 77.0 ppm) for
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Synthesis and Cytotoxicity of Piperazirum
1
13C NMR spectra. H NMR spectroscopic data is recorded as fol-
(m, 1 H), 1.00–0.86 (m, 12 H) ppm. 13C NMR (75 MHz, CDCl3):
lows: chemical shift {multiplicity, coupling constant(s) J [Hz], rela-
tive integral} where multiplicity is defined as: s = singlet; d = doub-
let; t = triplet; q = quartet; dd = doublet of doublets; dt = doublet
of triplets; m = multiplet; br. s = broad singlet; br. d = broad doub-
let. FTIR spectra were recorded with a Bruker (Alpha) spectrome-
ter. Optical rotation values were measured with a Horiba high-
sensitivity polarimeter using a 2 mL cell with a 10 mm path length.
Mass spectra were recorded with a Micromass VG-7070H mass
spectrometer using the ESI technique and are reported in mass
units (m/z). High-resolution mass spectra (HRMS, ESI) were ob-
tained using either a TOF or a double-focussing spectrometer.
δ = 156.2, 79.3, 78.9, 42.3, 30.9, 28.3, 24.7, 23.0, 22.2, 19.2,
18.3 ppm. IR (neat): ν = 3440, 2959, 2872, 1688, 1392, 1366, 1250,
˜
1171, 1047 cm–1. HRMS (ESI): calcd. for C14H30O3N [M + H]+
260.2220; found 260.2230.
Data for 6a: m.p. 78–80 °C. [α]2D6 = –36.4 (c = 0.38, CHCl3). 1H
NMR (300 MHz, CDCl3): δ = 4.66 (br. d, J = 9.0 Hz, 1 H), 3.75
(m, 1 H), 3.23 (m, 1 H), 1.90 (br. s, 1 H), 1.73–1.55 (m, 2 H), 1.43
(s, 9 H), 1.37–1.11 (m, 2 H), 1.03–0.87 (m, 12 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 155.7, 80.3, 79.0, 50.6, 36.9, 30.8, 28.3, 24.5,
23.9, 21.4, 19.2, 18.7 ppm. IR (neat): ν = 3369, 2956, 1683, 1531,
˜
1273, 1172 cm–1. HRMS (ESI): calcd. for C14H30O3N [M + H]+
260.2220; found 260.2234.
Methyl
(S)-2-(tert-Butoxycarbonylamino)-4-methylpentanoate
(5):[22] Thionyl chloride (5.56 mL, 76.231 mmol) was added drop-
wise to a solution of l-leucine (10 g, 76.231 mmol) in dry MeOH
(100 mL) at 0 °C under a nitrogen atmosphere. The resulting mix-
ture was stirred at room temp. for 2 h, and then it was heated at
reflux for 8 h. Then the reaction mixture was allowed to cool to
room temp., and the solvent was evaporated under reduced pres-
sure to give the methyl ester as a solid.
tert-Butyl [(3R,4S)-3-Azido-2,6-dimethylheptan-4-yl]carbamate (7):
Et3N (9.57 mL, 68.793 mmol) and methanesulfonyl chloride
(2.0 mL, 25.797 mmol) were added to a solution of alcohol 6
(4.42 g, 17.198 mmol) in CH2Cl2 (50 mL) under N2 at 0 °C. The
reaction was monitored by TLC. The reaction mixture was poured
into EtOAc, and the mixture was washed with water and brine. The
organic layer was dried with Na2SO4, filtered, and concentrated
in vacuo. Purification by flash column chromatography gave the
mesylate of 6 (4.5 g, 78%) as a yellow oil. Rf = 0.5 (15% EtOAc/
hexane). [α]2D6 = –31.9 (c = 0.54, CHCl3). 1H NMR (300 MHz,
CDCl3): δ = 4.48 (br. d, J = 10.5 Hz, 1 H), 4.35 (dd, J = 7.5, 3.0 Hz,
1 H), 4.05–3.93 (m, 1 H), 3.03 (s, 3 H), 2.00 (m, 1 H), 1.74–1.56
(m, 1 H), 1.46 (m, 1 H), 1.42 (s, 9 H), 1.39–1.20 (m, 1 H), 1.07–
0.99 (m, 6 H), 0.95 (dd, J = 6.0, 2.2 Hz, 6 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 155.3, 90.4, 79.2, 48.9, 38.6, 29.8, 28.2, 24.4,
The methyl ester was dissolved in dry CH2Cl2 (110 mL) under N2
at 0 °C, and Et3N (21.23 mL, 152.462 mmol) was added, followed
by Boc anhydride (19.24 mL, 83.854 mmol). The reaction mixture
was stirred at room temp. overnight. Water was added to the reac-
tion mixture, and the mixture was extracted with EtOAc. The or-
ganic layer was washed with brine, dried with Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
purified by flash column chromatography to give Boc-l-Leu-OMe
(5) (18.5 g, 97%) as a colourless oil. Rf = 0.5 (15% EtOAc/hexane).
1H NMR (300 MHz, CDCl3): δ = 4.85 (br. d, J = 8.3 Hz, 1 H),
4.25 (m, 1 H), 3.70 (s, 3 H), 1.68 (m, 1 H), 1.60–1.46 (m, 2 H), 1.41
(s, 9 H), 0.99–0.88 (m, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ
= 173.9, 155.3, 79.7, 52.1, 51.9, 41.7, 28.2, 24.7, 22.7, 21.8 ppm. IR
22.9, 21.8, 19.2, 17.9 ppm. IR (neat): ν = 3386, 2963, 2929, 1708,
˜
1511, 1340, 1170, 911 cm–1. HRMS (ESI): calcd. for C15H32O5NS
[M + H]+ 338.1996; found 338.1991.
The mesylate of 6 (4.5 g, 13.392 mmol) was dissolved in dry DMF
(20 mL), and sodium azide (1.74 g, 26.785 mmol) was added. The
mixture was stirred at 80 °C for 12 h. The reaction mixture was
cooled and poured in to ice-water. The mixture was extracted with
EtOAc, and the organic extract was washed with brine, dried with
Na2SO4, filtered, and concentrated in vacuo. Purification by flash
column chromatography gave azide 7 (3.16 g, 83%) as a white solid.
Rf = 0.5 (5% EtOAc/hexane), m.p. 85–87 °C. [α]2D6 = –22.3 (c =
0.65, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 4.39 (br. d, J =
10.5 Hz, 1 H), 3.95 (m, 1 H), 2.93 (dd, J = 9.0, 2.2 Hz, 1 H), 1.80
(m, 1 H), 1.64 (m, 1 H), 1.41 (s, 9 H), 1.36–1.19 (m, 2 H), 1.10–1.00
(m, 6 H), 0.99–0.92 (m, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ
= 155.2, 79.4, 74.1, 50.1, 37.2, 30.3, 28.3, 24.4, 23.8, 21.2, 20.2,
(neat): ν = 3366, 2960, 2873, 1760, 1715, 1514, 1366, 1251, 1167,
˜
1049 cm–1. HRMS (ESI): calcd. for C12H23O4NNa [M + Na]+
268.1519; found 268.1527.
Synthesis of 6 and 6a: DIBAL-H (1.0 m in toluene; 166.0 mL,
166.0 mmol) was added to
a solution of ester 5 (18.5 g,
75.454 mmol) in toluene at –78 °C under an argon atmosphere. The
reaction mixture was stirred for 10 min at that temperature, and
then the reaction was quenched by the addition of MeOH
(18.5 mL). The reaction mixture was then allowed to warm up to
room temp., and a saturated solution of sodium potassium tartrate
(85.24 g, 302.040 mmol) was added. The mixture was stirred for
1 h, and then the solids were removed by filtration through a bed
of Celite. The mixture was extracted with EtOAc, then the organic
phase was washed with brine, dried with Na2SO4, filtered, and con-
centrated under reduced pressure to give the aldehyde as a yellow
oil. This material was used in the next step without any further
purification. Rf = 0.4 (15% EtOAc/hexane).
19.0 ppm. IR (neat): ν = 3371, 2959, 2925, 2857, 2101, 1681, 1367,
˜
1255, 1169 cm–1. HRMS (ESI): calcd. for C14H28O2N4Na [M +
Na]+ 307.2104; found 307.2100.
tert-Butyl [(3R,4S)-3-Amino-2,6-dimethylheptan-4-yl]carbamate (3):
A solution of azide 7 (3.16 g, 11.126 mmol) in EtOAc/MeOH (2:1;
30 mL) was hydrogenated at balloon pressure (1 atm) in the pres-
ence of a catalytic amount of 10% Pd/C (450 mg). The reaction
was monitored by TLC. The mixture was then filtered through a
pad of Celite, which was then washed with EtOAc. The solvent was
evaporated from the filtrate, and the residue was purified by flash
chromatography to give amine 3 (2.64 g, 92%) as a colourless li-
Isopropylmagnesium
bromide
(1 m in
THF;
151 mL,
150.908 mmol) was added to a solution of aldehyde in THF
(150 mL) at 0 °C under a N2 atmosphere. The reaction was moni-
tored by TLC. The reaction was quenched with water, and the mix-
ture was extracted with EtOAc. The organic extract was washed
with brine, dried with Na2SO4, filtered, and concentrated in vacuo.
Purification by column chromatography gave a major product, 6
(12.7 g, 66.2%), as a pale yellow oil, and a minor product, 6a (3.6 g,
18.7%), as a white solid. Rf = 0.5 (15% EtOAc/hexane).
1
quid. Rf = 0.4 (EtOAc). [α]2D6 = –34.9 (c = 1.18, CHCl3). H NMR
(300 MHz, CDCl3): δ = 4.90 (br. d, J = 9.8 Hz, 1 H), 3.83–3.66 (m,
1 H), 2.27 (dd, J = 9.0, 4.5 Hz, 1 H), 1.73–1.58 (m, 1 H), 1.41 (s,
9 H), 1.26–1.03 (m, 3 H), 0.98–0.87 (m, 12 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 155.5, 78.4, 61.3, 49.8, 37.3, 31.7, 28.3, 24.5,
1
Data for 6: [α]2D6 = –22.3 (c = 0.65, CHCl3). H NMR (300 MHz,
CDCl3): δ = 4.68 (br. d, J = 9.8 Hz, 1 H), 3.76 (m, 1 H), 3.10 (m,
1 H), 2.16 (br. s, 1 H), 1.83–1.55 (m, 3 H), 1.42 (s, 9 H), 1.35–1.09
24.0, 21.4, 19.5 ppm. IR (neat): ν = 3343, 2961, 2926, 1706, 1368,
˜
Eur. J. Org. Chem. 2014, 1253–1265
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1257

G. Sudhakar, S. R. Bathula et al.
FULL PAPER
1251, 1170 cm–1. HRMS (ESI): calcd. for C14H31O2N2 [M + H]+
259.2380; found 259.2390.
(neat): ν = 3208, 2956, 1663, 1468, 1165, 1117 cm–1. HRMS (ESI):
˜
calcd. for C15H30ON2 [M + Na]+ 277.2255; found 277.2266.
tert-Butyl [(3S,4S)-3-Azido-2,6-dimethylheptan-4-yl]carbamate (7a):
The mesylate of 6a (994 g, 78%) was prepared as a yellow oil from
6a (975 mg, 3.793 mmol) following the procedure described for the
synthesis of the mesylate of 6. Rf = 0.5 (15 % EtOAc/hexane).
tert-Butyl {(3R,4S)-3-[(S)-2-Hydroxy-4-methylpentanamido]-2,6-di-
methylheptan-4-yl}carbamate (8): EDCI [1-ethyl-3-(dimethylamino-
propyl)carbodiimide] (3.92 g, 20.465 mmol) and HOBt (1-hy-
droxybenzotriazole; 2.762 g, 20.465 mmol) were added to a stirred
solution of acid (–)-4 (2.01 g, 15.348 mmol) in CH2Cl2 (20 mL) at
0 °C, and the reaction mixture was stirred for 10 min. Amine 3
(2.64 g, 10.235 mmol) in CH2Cl2 (11 mL) was added to this reac-
tion mixture, followed by DIPEA (8.91 mL, 51.162 mmol). Then
the reaction mixture was allowed to stir overnight. The reaction
mixture was diluted with EtOAc, and the mixture was washed with
HCl (1 n), saturated aq. NaHCO3, and brine. The organic layer
was dried with Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by column chromatography to
give peptide 8 (2.67 g, 70%) as a white solid. Rf = 0.5 (30% EtOAc/
hexane), m.p. 124–126 °C. [α]2D5.7 = –45 (c = 0.2, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 6.55 (br. d, J = 9.2 Hz, 1 H), 4.55 (br. d,
J = 8.8 Hz, 1 H), 4.16–4.05 (m, 1 H), 3.81–3.65 (m, 2 H), 3.27 (br.
s, 1 H), 1.94–1.76 (m, 2 H), 1.73–1.50 (m, 1 H), 1.43 (s, 9 H),
1.34–1.12 (m, 4 H), 1.02–0.83 (m, 18 H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 174.7, 155.7, 79.2, 70.8, 58.1, 50.2, 44.1, 40.4, 28.9,
[α]2D6 = –7.1 (c = 0.91, CHCl3). H NMR (500 MHz, CDCl3): δ =
1
4.83 (br. d, J = 8.0 Hz, 1 H), 4.54 (br. d, J = 8.0 Hz, 1 H), 3.93 (m,
1 H), 3.04 (s, 3 H), 1.90 (m, 1 H), 1.67 (m, 1 H), 1.42 (s, 9 H), 1.36
(m, 1 H), 1.23 (m, 1 H), 1.07–0.83 (m, 12 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 155.4, 91.3, 79.3, 49.3, 38.4, 37.0, 29.8, 28.3,
24.3, 23.7, 21.2, 19.5, 18.7 ppm. IR (neat): ν = 3388, 2964, 2927,
˜
1708, 1513, 1339, 1170, 912 cm–1. HRMS (ESI): calcd. for
C15H32O5NS [M + H]+ 338.1996; found 338.1998.
Azide 7a (647 mg, 77%) was prepared as a colourless liquid from
the mesylate of 6a (994 mg, 2.958 mmol) following the procedure
described for the synthesis of 7. Rf = 0.5 (5% EtOAc/hexane).
1
[α]2D6 = –45.2 (c = 0.37, CHCl3). H NMR (300 MHz, CDCl3): δ =
4.40 (br. d, J = 9.8 Hz, 1 H), 3.94 (m, 1 H), 2.93 (dd, J = 9.0,
2.2 Hz, 1 H), 1.80 (m, 1 H), 1.63 (m, 1 H), 1.41 (s, 9 H), 1.33–1.18
(m, 2 H), 1.08–0.99 (m, 6 H), 0.98–0.92 (m, 6 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 155.4, 79.1, 74.0, 49.3, 43.6, 30.8, 28.3, 24.8,
28.3, 24.6, 24.5, 23.8, 23.5, 21.5, 21.4, 20.3, 17.8 ppm. IR (neat): ν
˜
22.9, 22.3, 19.7, 19.6 ppm. IR (neat): ν = 3392, 2961, 2925, 2099,
˜
= 3447, 2922, 2852, 1654, 1369, 1170 cm–1. HRMS (ESI): calcd.
for C20H41O4N2 [M + H]+ 373.3060; found 373.3078.
1646, 1166 cm–1. HRMS (ESI): calcd. for C14H28O2N4Na [M +
Na]+ 307.2104; found 307.2100.
(S)-1-[(3R,4S)-4-(tert-Butoxycarbonylamino)-2,6-dimethylheptan-
3-ylamino]-4-methyl-1-oxopentan-2-yl Methanesulfonate (9): Et3N
(3.99 mL, 28.709 mmol) and methanesulfonyl chloride (0.83 mL,
10.766 mmol) were added to a solution of alcohol 8 (2.67 g,
7.177 mmol) in CH2Cl2 (22 mL) under N2 at 0 °C. The reaction
was monitored by TLC. The reaction was quenched with water,
and the mixture was extracted with EtOAc. The organic extract
was washed with brine, dried with Na2SO4, filtered, and concen-
trated under vacuum. Purification by flash column chromatog-
raphy gave mesylate 9 (3.13 g, 97%) as a white solid. Rf = 0.5 (30%
EtOAc/hexane), m.p. 162–164 °C. [α]2D6 = –61.4 (c = 0.24, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 6.30 (br. d, J = 9.0 Hz, 1 H),
4.95 (dd, J = 9.0, 3.7 Hz, 1 H), 4.48 (br. d, J = 8.6 Hz, 1 H), 3.86–
3.72 (m, 2 H), 3.11 (s, 3 H), 1.89–1.62 (m, 4 H), 1.44 (s, 9 H),
1.31–1.14 (m, 3 H), 1.05–0.88 (m, 18 H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 168.8, 155.6, 79.1, 78.8, 58.3, 50.3, 41.2, 39.8, 38.7,
28.8, 28.2, 24.6, 24.1, 23.6, 22.9, 21.5, 21.3, 20.0, 17.9 ppm. IR
tert-Butyl [(3S,4S)-3-Amino-2,6-dimethylheptan-4-yl]carbamate
(3a): Compound 3a (547 mg, 93%) was prepared as a colourless
liquid from azide 7a (647 mg, 2.278 mmol) following the procedure
described for the synthesis of 3. Rf = 0.4 (EtOAc). [α]2D6 = –33.3 (c
1
= 0.48, CHCl3). H NMR (300 MHz, CDCl3): δ = 4.81 (br. d, J =
9.0 Hz, 1 H), 3.75 (m, 1 H), 2.33 (m, 1 H), 1.70–1.50 (m, 2 H), 1.41
(s, 9 H), 1.33 (m, 1 H), 1.14 (m, 1 H), 0.98–0.88 (m, 12 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 155.8, 78.4, 59.8, 49.4, 43.2, 30.6,
28.2, 24.7, 22.9, 22.2, 19.8, 18.6 ppm. IR (neat): ν = 3362, 2960,
˜
1646, 1250, 1171 cm–1. HRMS (ESI): calcd. for C14H31O2N2 [M +
H]+ 259.2380; found 259.2395.
tert-Butyl [(3S,4S)-3-[(S)-2-Hydroxy-4-methylpentanamido]-2,6-di-
methylheptan-4-yl]carbamate (8a): Compound 8a (513 mg, 65%)
was prepared as a white solid from the coupling of acid (–)-4
(417 mg, 3.180 mmol) and amine 3a (547 mg, 2.120 mmol) follow-
ing the procedure described for the synthesis of 8. Rf = 0.5 (30%
EtOAc/hexane), m.p. 140–142 °C. [α]2D6 = –85.2 (c = 0.085, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 6.76 (br. d, J = 9.8 Hz, 1 H),
5.78 (br. d, J = 9.0 Hz, 0.4 H), 4.68 (br. d, J = 8.3 Hz, 0.6 H), 4.12
(m, 1 H), 3.80–3.48 (m, 2 H), 1.96–1.79 (m, 2 H), 1.73–1.49 (m, 2
H), 1.40 (s, 9 H), 1.31–1.14 (m, 4 H), 0.99–0.78 (m, 18 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 175.2, 156.8, 79.3, 71.0, 58.3,
57.9, 50.6, 43.6, 41.5, 28.7, 28.3, 24.5, 23.5, 21.5, 21.2 ppm. IR
(neat): ν = 3208, 2956, 2870, 1663, 1468 cm–1. HRMS (ESI): calcd.
˜
for C21H43O6N2S [M + H]+ 451.2836; found 451.2855.
(3R,5S,6R)-3,5-Diisobutyl-6-isopropylpiperazin-2-one (1): TFA
(1.75 mL) was added slowly in a dropwise manner to a solution of
9 (1.1 g, 2.444 mmol) in CH2Cl2 (7 mL) under N2 at 0 °C. The reac-
tion was monitored by TLC. After the reaction was complete, the
solvent was evaporated under vacuum to give the amine, which was
used in the next reaction without further purification.
(neat): ν = 3360, 2957, 2922, 2853, 1741, 1647, 1250, 1171, 1091,
˜
1044 cm–1. HRMS (ESI): calcd. for C20H41O4N2 [M + H]+
373.3060; found 373.3080.
The amine was dissolved in dry MeOH (49 mL), and diisopropyl-
ethylamine (DIPEA; 1.27 mL, 7.333 mmol) was added. The reac-
tion mixture was heated at reflux overnight. After the reaction was
complete, the solvent was evaporated under reduced pressure, and
the residue was purified by column chromatography to give cyclic
compound 1 (509 mg, 82%) as a white solid. Rf = 0.5 (30% EtOAc/
hexanes), m.p. 53–55 °C. [α]2D6 = +52.5 (c = 0.33, MeOH). 1H NMR
(300 MHz, CDCl3): δ = 6.21 (br. s, 1 H), 3.38 (dd, J = 9.8, 3.7 Hz,
1 H), 3.15 (m, 1 H), 3.07 (dd, J = 6.7, 3.7 Hz, 1 H), 1.99–1.82 (m,
(S)-1-[(3S,4S)-4-(tert-Butoxycarbonylamino)-2,6-dimethylheptan-
3-ylamino]-4-methyl-1-oxopentan-2-yl Methanesulfonate (9a): Mes-
ylate 9a (485 mg, 78%) was prepared as a colourless liquid from
8a (513 g, 1.379 mmol) following the procedure described for the
synthesis of 9. Rf = 0.5 (30% EtOAc/hexane). [α]2D6 = –89.8 (c =
0.57, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 6.49 (br. d, J =
7.9 Hz, 1 H), 5.09–5.03 (m, 1 H), 4.71 (br. d, J = 8.9 Hz, 1 H), 3.90
(br. s, 1 H), 3.62 (m, 1 H), 3.15 (s, 3 H), 1.84–1.71 (m, 4 H), 1.67–
2 H), 1.81–1.59 (m, 1 H), 1.44–1.22 (m, 4 H), 1.02–0.80 (m, 18 H) 1.58 (m, 1 H), 1.43 (s, 9 H), 1.37–1.28 (m, 2 H), 1.08–0.86 (m, 18
ppm. 13C NMR (75 MHz, CDCl3): δ = 174.2, 59.3, 56.8, 53.3, 41.1, H) ppm. 13C NMR (75 MHz, CDCl3): δ = 168.8, 156.1, 79.0, 58.3,
40.4, 27.8, 24.8, 24.4, 23.6, 23.0, 22.4, 21.5, 20.9, 17.7 ppm. IR 50.2, 49.7, 42.1, 41.3, 38.7, 29.7, 28.3, 24.6, 24.4, 23.1, 22.9, 22.0,
1258
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 1253–1265

Synthesis and Cytotoxicity of Piperazirum
20.9, 20.1, 18.8 ppm. IR (neat): ν = 3389, 2958, 2923, 2855, 1677, (br. s, 1 H), 1.95–1.73 (m, 4 H), 1.71–1.48 (m, 1 H), 1.41 (s, 9 H),
˜
1646, 1360, 1171, 1092 cm– 1 . HRMS (ESI): calcd. for
C21H42O6N2NaS [M + Na]+ 473.2655; found 473.2655.
1.33–1.22 (m, 2 H), 1.01–0.84 (m, 18 H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 175.5, 156.6, 79.2, 70.5, 58.1, 50.1, 44.2, 42.0, 28.9,
28.3, 24.7, 24.4, 23.5, 21.5, 21.1, 20.4 ppm. IR (neat): ν = 3332,
˜
(3R,5S,6S)-3,5-Diisobutyl-6-isopropylpiperazin-2-one (1a): Cyclic
compound 1a (84 mg, 74%) was prepared as an amorphous solid
from 9a (200 mg, 0.444 mmol) following the procedure described
for the synthesis of 1. Rf = 0.4 (50% EtOAc/hexane). [α]2D6 = –61.5
2957, 2922, 2855, 1740, 1690, 1367, 1170, 1091 cm–1. HRMS (ESI):
calcd. for C20H41O4N2 [M + H]+ 373.3060; found 373.3059.
(R)-1-[(3S,4S)-4-(tert-Butoxycarbonylamino)-2,6-dimethylheptan-
3-ylamino]-4-methyl-1-oxopentan-2-yl Methanesulfonate (9c): Mes-
ylate 9c (333 mg, 72 %) was prepared as a white solid from 8c
(382 mg, 1.027 mmol) following the procedure described for the
synthesis of 9. Rf = 0.5 (30% EtOAc/hexane), m.p. 94–96 °C. [α]2D6
= +2.26 (c = 0.77, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 6.74
(br. d, J = 8.0 Hz, 1 H), 5.04 (m, 1 H), 4.51 (br. d, J = 7.0 Hz, 1
H), 3.76–3.63 (m, 2 H), 3.13 (s, 3 H), 1.94–1.60 (m, 5 H), 1.42 (s,
9 H), 1.35–1.27 (m, 2 H), 1.04–0.86 (m, 18 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 169.4, 156.4, 78.2, 58.4, 50.2, 41.8, 41.4,
38.6, 29.2, 28.3, 24.7, 24.2, 23.3, 23.1, 21.6, 21.0, 20.2 ppm. IR
1
(c = 0.065, MeOH). H NMR (300 MHz, CDCl3): δ = 5.72 (br. s,
1 H), 3.43 (dd, J = 10.3, 3.9 Hz, 1 H), 2.93–2.84 (m, 2 H), 1.99–
1.73 (m, 2 H), 1.71–1.48 (m, 4 H), 1.35–1.27 (m, 1 H), 0.99–0.85
(m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 174.6, 63.4, 53.5,
46.8, 41.1, 40.6, 29.5, 24.5, 24.4, 23.6, 23.5, 21.5, 21.0, 19.7,
16.0 ppm. IR (neat): ν = 3200, 2956, 2927, 1693, 1315 cm–1. HRMS
˜
(ESI): calcd. for C15H31ON2 [M + H]+ 255.2430; found 255.2433.
tert-Butyl [(3R,4S)-3-[(R)-2-Hydroxy-4-methylpentanamido]-2,6-di-
methylheptan-4-yl]carbamate (8b): Compound 8b (1.0 g, 52%) was
prepared as a white solid from the coupling of acid (+)-4 (1.0 g,
7.674 mmol) and amine 3 (1.32 g, 5.116 mmol) following the pro-
cedure described for the synthesis of 8. Rf = 0.5 (30% EtOAc/hex-
ane), m.p. 58–60 °C. [α]2D6 = –35.6 (c = 0.07, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 6.53 (br. d, J = 10.5 Hz, 0.5 H), 6.06 (br.
d, J = 8.3 Hz, 0.5 H), 4.57 (m, 1 H), 4.18–4.06 (m, 1 H), 3.86–3.66
(m, 2 H), 1.96–1.56 (m, 3 H), 1.45 (m, 9 H), 1.32–1.05 (m, 4 H),
1.01–0.83 (m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 174.8,
155.9, 79.3, 71.1, 57.9, 50.5, 43.9, 40.4, 28.9, 28.3, 24.7, 24.6, 23.7,
(neat): ν = 3360, 2959, 2925, 2860, 1741, 1684, 1362, 1248,
˜
1172 cm–1. HRMS (ESI): calcd. for C21H43O6N2S [M + H]+
451.2836; found 451.2834.
(3S,5S,6S)-3,5-Diisobutyl-6-isopropylpiperazin-2-one (1c): Cyclic
compound 1c (60 mg, 71%) was prepared as a white solid from
9c (150 mg, 0.333 mmol) following the procedure described for the
synthesis of 1. Rf = 0.4 (50% EtOAc/hexane), m.p. 74–76 °C. [α]2D6
1
= –59.8 (c = 0.29, MeOH). H NMR (300 MHz, CDCl3): δ = 6.01
23.4, 21.6, 21.3, 20.4 ppm. IR (neat): ν = 3391, 2957, 2922, 2853,
˜
(br. s, 1 H), 3.41 (dd, J = 10.1, 4.1 Hz, 1 H), 2.94–2.82 (m, 2 H),
1.98–1.72 (m, 3 H), 1.69–1.47 (m, 2 H), 1.36–1.21 (m, 2 H), 1.01–
0.85 (m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 174.5, 63.2,
53.3, 46.7, 41.0, 40.5, 29.5, 24.4, 24.3, 23.5, 23.4, 21.5, 20.9, 19.6,
1705, 1647, 1367, 1171, 1079 cm–1. HRMS (ESI): calcd. for
C20H41O4N2 [M + H]+ 373.3060; found 373.3068.
(R)-1-[(3R,4S)-4-(tert-Butoxycarbonylamino)-2,6-dimethylheptan-
3-ylamino]-4-methyl-1-oxopentan-2-yl Methanesulfonate (9b): Mes-
ylate 9b (992 mg, 82 %) was prepared as a white solid from 8b
(1.0 g, 2.688 mmol) following the procedure described for the syn-
thesis of 9. Rf = 0.5 (30% EtOAc/hexane), m.p. 144–146 °C. [α]2D6
= –5.0 (c = 0.1, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 6.31
(br. d, J = 9.8 Hz, 1 H), 5.00 (m, 1 H), 4.61 (br. d, J = 9.0 Hz, 1
H), 3.91–3.77 (m, 2 H), 3.13 (s, 3 H), 1.90–1.56 (m, 5 H), 1.42 (s,
9 H), 1.30–1.13 (m, 2 H), 1.05–0.85 (m, 18 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 168.9, 155.8, 79.3, 58.4, 50.0, 41.5, 38.8,
38.3, 29.4, 28.3, 24.6, 24.4, 23.7, 23.1, 21.4, 21.1, 19.9, 18.7 ppm.
16.0 ppm. IR (neat): ν = 3207, 2954, 2929, 1649, 1667 cm–1. HRMS
˜
(ESI): calcd. for C15H31ON2 [M + H]+ 255.2430; found 255.2435.
Methyl (S)-2-(tert-Butoxycarbonylamino)-3-methylbutanoate
(10):[26] Compound 10 (9.1 g, 98%) was prepared as a colourless
oil from l-valine (5 g, 42.680 mmol) following the procedure de-
scribed for the synthesis of 5. Rf = 0.5 (15% EtOAc/hexane). 1H
NMR (300 MHz, CDCl3): δ = 5.01 (br. d, J = 9.0 Hz, 1 H), 4.22
(dd, J = 9.0, 4.5 Hz, 1 H), 3.73 (s, 3 H), 2.11 (m, 1 H), 1.44 (s, 9
H), 0.95 (d, J = 6.7 Hz, 3 H), 0.88 (d, J = 6.7 Hz, 3 H) ppm. 13C
NMR (75 MHz, CDCl3): δ = 172.7, 155.5, 79.5, 58.4, 51.8, 31.1,
IR (neat): ν = 3366, 2957, 2922, 2853, 1741, 1648, 1395, 1177 cm–1.
˜
28.1, 18.8, 17.5 ppm. IR (neat): ν = 3342, 2961, 2921, 1760, 1714,
˜
HRMS (ESI): calcd. for C21H42O6N2NaS [M + H]+ 473.2655;
found 473.2658.
1503, 1243, 1170 cm–1. HRMS (ESI): calcd. for C11H21O4NNa [M
+ Na]+ 254.1367; found 254.1369.
(3S,5S,6R)-3,5-Diisobutyl-6-isopropylpiperazin-2-one (1b): Cyclic
compound 1b (23 mg, 64%) was prepared as a colourless liquid
from 9b (100 mg, 0.222 mmol) following the procedure described
for the synthesis of 1. Rf = 0.5 (30% EtOAc/hexane). [α]2D6 = –73.5
tert-Butyl [(3S)-4-Hydroxy-2,6-dimethylheptan-3-yl]carbamate (11):
A diastereomeric mixture of alcohols 11 (dr = 7:1; 8.68 g, 80%)
was prepared as a yellow oil by the reaction of isobutylmagnesium
bromide (1 m in THF; 83.8 mL, 83.8 mmol) with the aldehyde de-
rived from 10 (9.1 g, 41.900 mmol) following the procedure de-
scribed for the synthesis of 6 and 6a. Rf = 0.5 (15% EtOAc/hexane).
[α]2D6 = +40.2 (c = 0.23, CHCl3). 1H NMR (300 MHz, CDCl3): δ =
4.71 (br. d, J = 9.8 Hz, 1 H), 3.83 (m, 1 H), 3.11 (m, 1 H), 1.90–
1.68 (m, 2 H), 1.43 (s, 9 H), 1.36–1.21 (m, 2 H), 1.02–0.86 (m, 12
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 156.7, 78.8, 68.9, 59.9,
1
(c = 0.06, MeOH). H NMR (500 MHz, CDCl3): δ = 6.23 (br. s, 1
H), 3.43–3.34 (m, 2 H), 2.57 (dd, 10.4, 3.1, 1 H), 1.87 (m, 1 H),
1.79 (m, 1 H), 1.69 (m, 1 H), 1.61 (m, 1 H), 1.46 (m, 1 H), 1.29
(m, 1 H), 1.11 (m, 1 H), 1.03–0.83 (m, 18 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 173.6, 62.6, 57.6, 51.5, 41.0, 38.4, 28.5, 24.4,
24.2, 23.8, 23.5, 20.9, 20.8, 20.2, 19.0 ppm. IR (neat): ν = 3207,
˜
2956, 2926, 1693, 1315 cm–1. HRMS (ESI): calcd. for C15H31ON2
[M + H]+ 255.2430; found 255.2432.
43.8, 30.0, 28.3, 24.3, 23.1, 22.2, 19.9, 19.1 ppm. IR (neat): ν =
˜
3394, 2957, 2922, 2854, 1689, 1245, 1169 cm–1. HRMS (ESI): calcd.
for C14H30O3N [M + H]+ 260.2220; found 260.2228.
tert-Butyl [(3S,4S)-3-[(R)-2-Hydroxy-4-methylpentanamido]-2,6-di-
methylheptan-4-yl]carbamate (8c): Compound 8c (382 mg, 53 %)
was prepared as a white solid from the coupling of acid (+)-4
(381 mg, 2.906 mmol) and amine 3a (500 mg, 1.937 mmol) follow-
ing the procedure described for the synthesis of 8. Rf = 0.5 (30%
Benzyl [(2S)-1-[(3S)-4-Hydroxy-2,6-dimethylheptan-3-ylamino]-4-
methyl-1-oxopentan-2-yl]carbamate (13): TFA (16 mL) was added
slowly in a dropwise manner to a solution of compound 11 (5.62 g,
21.698 mmol) in CH2Cl2 (65 mL) under N2 at 0 °C. The reaction
was monitored by TLC. After the reaction was complete, the sol-
vent was evaporated to give the free amine.
1
EtOAc/hexane), m.p. 108–110 °C. [α]2D6 = –40 (c = 0.2, CHCl3). H
NMR (300 MHz, CDCl3): δ = 6.60 (br. d, J = 9.8 Hz, 1 H), 4.54
(br. d, J = 8.3 Hz, 1 H), 4.12 (m, 1 H), 3.79–3.58 (m, 2 H), 3.01
Eur. J. Org. Chem. 2014, 1253–1265
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1259

G. Sudhakar, S. R. Bathula et al.
FULL PAPER
Compound 12 (Cbz-l-Leu-OH)[27] (740 mg, 2.825 mmol) was dis-
DMAP (catalytic amount) were added to a solution of compound
solved in CH2Cl2 (2 mL), and EDCI (722.16 mg, 3.767 mmol) and (–)-1 (34 mg, 0.1338 mmol) in CH2Cl2 (1.5 mL) at 0 °C, and the
HOBt (508 mg, 3.767 mmol) were added at 0 °C. The reaction mix-
ture was stirred for 10 min. Then the free amine (300 mg,
1.883 mmol) prepared above was added in CH2Cl2 (4 mL), and
then DIPEA (1.64 mL, 9.415 mmol) was added. The reaction mix-
ture was allowed to stir overnight. The reaction mixture was diluted
with EtOAc, and this mixture was washed with HCl (1 n), saturated
aq. NaHCO3, and brine. The organic layer was dried with Na2SO4,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography to give peptide 13 (512 mg,
67%). [α]2D6 = –22.7 (c = 0.33, CHCl3). 1H NMR (500 MHz,
CDCl3): δ = 7.33–7.22 (m, 5 H), 6.61 (br. d, J = 8.4 Hz, 1 H), 5.43
reaction mixture was allowed to stir for 30 min. After the reaction
was complete, H2O was added, and the mixture was stirred for a
further 10 min. The mixture was extracted with EtOAc, and the
organic phase was washed with water and brine, dried with
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography to give acetylated
compound 15 (31 mg, 79%). 1H NMR (500 MHz, CDCl3): δ = 5.96
(br. s, 1 H), 5.17 (d, J = 13.2 Hz, 0.4 H), 5.03 (t, J = 6.6 Hz, 0.6
H), 4.22 (dd, J = 8.8, 4.4 Hz, 0.4 H), 3.98 (d, J = 12.1 Hz, 0.6 H),
3.06 (dd, J = 11.0, 3.3 Hz, 0.6 H), 3.00 (dd, J = 9.9, 3.3 Hz, 0.4
H), 2.26 (m, 0.5 H), 2.15 (s, 3 H), 2.04 (m, 0.5 H), 1.92 (m, 1 H),
(br. d, J = 8.4 Hz, 1 H), 5.12–5.00 (m, 2 H), 4.19–4.10 (m, 1 H), 1.70–1.54 (m, 3 H), 1.34 (m, 1 H), 1.10 (m, 1 H), 1.05–0.93 (m, 18
3.88 (m, 1 H), 3.45 (m, 1 H), 1.89–1.78 (m, 1 H), 1.75–1.58 (m, 4 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 171.7, 170.5, 169.9,
H), 1.48 (m, 1 H), 1.17 (m, 1 H), 1.00–0.80 (m, 18 H) ppm. 13C 169.5, 62.6, 61.5, 54.8, 52.8, 52.0, 46.4, 45.5, 43.9, 33.7, 29.2, 29.1,
NMR (75 MHz, CDCl3): δ = 172.4, 156.2, 136.1, 128.4, 128.0, 25.7, 25.5, 23.8, 23.6, 23.5, 23.4, 23.2, 22.5, 22.3, 22.2, 22.0, 21.7,
127.9, 68.3, 66.9, 58.6, 53.9, 43.9, 41.2, 29.7, 24.6, 24.3, 23.0, 22.6, 21.5, 21.4, 19.5, 18.0, 17.9 ppm. IR (neat): ν = 3441, 2925, 2858,
˜
22.2, 19.7, 19.2 ppm. IR (neat): ν = 3315, 2956, 2929, 2871, 1694, 1667, 1631, 1325, 1115 cm–1. HRMS (ESI): calcd. for C17H33O2N2
˜
1647, 1531, 1462, 1257, 1120, 1042 cm–1. HRMS (ESI): calcd. for [M + H]+ 297.2536; found 297.2547.
C23H39O4N2 [M + H]+ 407.2904; found 407.2920.
Benzyl [2-[(3S)-4-Hydroxy-2,6-dimethylheptan-3-ylamino]-2-oxo-
Benzyl [(S)-1-[(S)-2,6-Dimethyl-4-oxoheptan-3-ylamino]-4-methyl-1- ethyl]carbamate (16a): EDCI (192.7 mg, 1.006 mmol) and HOBt
oxopentan-2-yl]carbamate (14): Dess–Martin periodinane (DMP;
641.6 mg, 1.513 mmol) was added to a stirred solution of alcohol
13 (512 mg, 1.261 mmol) in CH2Cl2 (4 mL) at 0 °C. The resulting
solution was stirred at room temp., and the reaction was monitored
by TLC. The reaction was quenched with a mixture of saturated
aq. NaHCO3 and saturated aq. Na2S2O3 (1:1). The resulting mix-
ture was stirred for 15 min to give a clear solution. The aqueous
layer was extracted with EtOAc. The organic layer was washed with
brine, dried with Na2SO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash column chromatography
to give ketone 14 (450 mg, 88%) as a white solid. Rf = 0.5 (30%
EtOAc/hexane), m.p. 80–82 °C. [α]2D6 = +22.1 (c = 0.52, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 7.34–7.24 (m, 5 H), 6.52 (br. d,
J = 7.8 Hz, 1 H), 5.20 (br. d, J = 7.8 Hz, 1 H), 5.12–5.04 (m, 2 H),
4.51 (dd, J = 8.7, 3.9 Hz, 1 H), 4.18 (m, 1 H), 2.44–2.27 (m, 2 H),
2.23–2.10 (m, 2 H), 1.71–1.57 (m, 2 H), 1.49 (m, 1 H), 0.98–0.87
(135.8 mg, 1.006 mmol) were added to a stirred solution of acid
12a (Cbz-Gly-OH; 157.7 mg, 0.7547 mmol) in CH2Cl2 (2 mL) at
0 °C, and the reaction mixture was stirred for 10 min. The free
amine derived from 11 (TFA, CH2Cl2; 80 mg, 0.503 mmol) was
added in CH2 Cl2 (3 mL), followed by DIPEA (0.43 mL,
2.5157 mmol). The reaction mixture was allowed to stir overnight.
The reaction mixture was diluted with EtOAc, and this mixture
was washed with HCl (1 n), saturated aq. NaHCO3, and brine. The
organic layer was dried with Na2SO4, filtered, and concentrated
under reduced pressure. The residue was purified by column
chromatography to give peptide 16a (113 mg, 64%). Rf = 0.4 (60%
EtOAc/hexane). [α]2D6 = –14.5 (c = 0.84, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 7.42–7.25 (m, 5 H), 6.13 (br. d, J = 9.6 Hz,
1 H), 5.76 (br. s, 1 H), 5.48 (br. s, 1 H), 5.20–5.01 (m, 3 H), 4.00–
3.72 (m, 2 H), 1.72 (m, 1 H), 1.55 (m, 1 H), 1.42–1.22 (m, 2 H),
0.98–0.77 (m, 12 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 170.2,
(m, 16 H), 0.79–0.72 (m, 3 H) ppm. 13C NMR (75 MHz, CDCl3): 169.6, 156.7, 136.0, 128.4, 128.0, 127.9, 73.0, 67.1, 67.0, 56.8, 44.6,
δ = 208.4, 172.5, 156.0, 136.1, 128.2, 127.8, 127.7, 66.7, 62.4, 53.4,
42.6, 40.5, 29.1, 24.3, 23.0, 21.7, 19.9 ppm. IR (neat): ν = 3333,
˜
49.7, 41.2, 29.8, 24.4, 23.9, 22.6, 22.4, 22.2, 21.9, 19.7, 16.6 ppm.
2958, 2924, 2855, 1707, 1676, 1518, 1461, 1392, 1265, 1199,
IR (neat): ν = 3304, 2958, 2926, 1706, 1695, 1532, 1462, 1122, 1052 cm–1. HRMS (ESI): calcd. for C19H31O4N2 [M + H]+
˜
1043 cm–1. HRMS (ESI): calcd. for C23H37O4N2 [M + H]+
405.2753; found 405.2761.
351.2283; found 351.2274.
Benzyl [(2S)-1-[(3S)-4-Hydroxy-2,6-dimethylheptan-3-ylamino]-1-
oxopropan-2-yl]carbamate (16b): Compound 16b (125 mg, 54%)
was prepared as a colourless oil from the coupling of 12b (Cbz-l-
Ala-OH)[29] (210 mg, 0.9433 mmol) and the free amine derived
from 11 (TFA, CH2Cl2; 100 mg, 0.628 mmol) following the pro-
cedure described for the synthesis of 16a. Rf = 0.4 (50% EtOAc/
hexane). [α]2D6 = –65.3 (c = 0.13, CHCl3). 1H NMR (300 MHz,
CDCl3): δ = 7.40–7.30 (m, 5 H), 6.35 (br. d, J = 8.8 Hz, 1 H), 5.48
(br. d, J = 7.1 Hz, 1 H), 5.18–5.05 (m, 2 H), 4.25 (m, 1 H), 3.90
(m, 1 H), 3.49 (m, 1 H), 1.96–1.60 (m, 3 H), 1.51–1.36 (m, 3 H),
1.35–1.11 (m, 1 H), 1.07–0.81 (m, 12 H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 172.4, 155.8, 136.1, 128.5, 128.1, 128.0, 68.6, 66.9,
(3S,5R,6S)-3,5-Diisobutyl-6-isopropylpiperazin-2-one (–)-1: A solu-
tion of ketone 14 (200 mg, 0.495 mmol) in MeOH (4 mL) was hy-
drogenated at balloon pressure (1 atm) in the presence of 10% Pd/
C (200 mg). The reaction was monitored by TLC. The mixture was
filtered through a pad of Celite, which was then washed with
EtOAc. The solvent was evaporated from the filtrate under reduced
pressure, and the residue was purified by column chromatography
to give cyclic compound (–)-1 (94 mg, 74%) as a white solid, m.p.
53–55 °C. [α]2D6 = –55.2 (c = 0.65, MeOH). 1H NMR (300 MHz,
CDCl3): δ = 6.66 (br. s, 1 H), 3.36 (dd, J = 9.8, 3.7 Hz, 1 H), 3.15
(m, 1 H), 3.06 (dd, J = 6.7, 3.7 Hz, 1 H), 1.95–1.82 (m, 1 H), 1.81–
1.60 (m, 1 H), 1.42–1.24 (m, 5 H), 1.04–0.88 (m, 18 H) ppm. 13C
NMR (75 MHz, CDCl3): δ = 174.4, 59.2, 56.8, 53.2, 41.1, 40.4,
58.5, 50.8, 43.8, 29.8, 24.4, 23.1, 22.2, 19.7, 19.2 ppm. IR (neat): ν
˜
= 3393, 2957, 2925, 1740, 1694, 1516, 1248, 1049 cm–1. HRMS
(ESI): calcd. for C20H33O4N2 [M + H]+ 365.2440; found 365.2445.
27.8, 24.7, 24.3, 23.5, 23.0, 22.4, 21.5, 20.9, 17.7 ppm. IR (neat): ν
˜
= 3343, 2957, 2871, 1663, 1469, 1280, 1164 cm–1. HRMS (ESI):
calcd. for C15H31ON2 [M + H]+ 255.2430; found 255.2440.
Benzyl [(2S)-1-[(3S)-4-Hydroxy-2,6-dimethylheptan-3-ylamino]-3-
methyl-1-oxobutan-2-yl]carbamate (16c): Compound 16c (142 mg,
(3S,5R,6S)-4-Acetyl-3,5-diisobutyl-6-isopropylpiperazin-2-one (15): 57%) was prepared as a colourless liquid from the coupling of 12c
Ac2O (0.16 mL, 0.1338 mmol), Et3N (0.022 mL, 0.1606 mmol), and (Cbz-l-Val-OH; 236.7 mg, 0.9433 mmol) and the free amine de-
1260
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© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 1253–1265

Synthesis and Cytotoxicity of Piperazirum
rived from 11 (100 mg, 0.6289 mmol) following the procedure de-
scribed for the synthesis of 16a. Rf = 0.4 (30% EtOAc/hexane). [α]
was prepared as a pale yellow oil from the coupling of 12b (Cbz-
l-Ala-OH; 210 mg, 0.9433 mmol) and the free amine derived from
6 and 6a (100 mg, 0.6289 mmol) following the procedure described
= –26.6 (c = 0.26, CHCl3). 1H NMR (300 MHz, CDCl3): δ =
26
D
7.40–7.29 (m, 5 H), 6.28 (br. d, J = 8.8 Hz, 1 H), 5.38 (br. d, J =
for the synthesis of 16a. Rf = 0.4 (50% EtOAc/hexane). [α]2D5.7
=
8.6 Hz, 1 H), 5.18–5.04 (dd, J = 17.9, 12.2 Hz, 2 H), 3.98 (dd, J = +5.2 (c = 0.33, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 7.42–
8.8, 6.2 Hz, 1 H), 3.91 (m, 1 H), 3.56–3.47 (m, 1 H), 2.25–1.97 (m,
7.28 (m, 5 H), 6.15 (br. d, J = 8.3 Hz, 1 H), 5.39 (br. s, 1 H), 5.17–
2 H), 1.86 (m, 1 H), 1.39–1.15 (m, 2 H), 1.04–0.82 (m, 18 H) ppm. 5.03 (m, 2 H), 4.27–4.06 (m, 2 H), 3.15 (m, 1 H), 2.15 (m, 1 H),
13C NMR (125 MHz, CDCl3): δ = 171.5, 156.5, 136.2, 128.4, 128.0, 1.65–1.44 (m, 3 H), 1.43–1.24 (m, 4 H), 1.02–0.84 (m, 12 H) ppm.
127.8, 68.4, 66.9, 61.0, 58.5, 44.1, 30.5, 29.9, 24.3, 22.9, 22.3, 19.7, 13C NMR (75 MHz, CDCl3): δ = 172.1, 155.8, 136.1, 128.4, 128.0,
19.4, 17.8 ppm. IR (neat): ν = 3391, 2925, 1694, 1647, 1514, 1171,
127.8, 78.7, 66.8, 62.5, 50.6, 48.7, 41.9, 31.1, 24.6, 22.9, 22.3, 19.0,
˜
1094 cm–1. HRMS (ESI): calcd. for C22H37O4N2 [M + H]+
393.2753; found 393.2761.
18.7, 18.2 ppm. IR (neat): ν = 3392, 2956, 2926, 1740, 1648, 1517,
˜
1462, 1367, 1249, 1063 cm–1. HRMS (ESI): calcd. for C20H33O4N2
[M + H]+ 365.2440; found 365.2444.
Benzyl [(2S,3S)-1-[(3S)-4-Hydroxy-2,6-dimethylheptan-3-ylamino]-
3-methyl-1-oxopentan-2-yl]carbamate (16d): Compound 16d
(158 mg, 62%) was prepared as a solid from the coupling of 12d
(Cbz-l-Ileu-OH)[30] (249.9 mg, 0.9433 mmol) with the free amine
derived from 11 (100 mg, 0.6289 mmol) following the procedure
described for the synthesis of 16a. Rf = 0.35 (30% EtOAc/hexane),
m.p. 110–112 °C. [α]2D6 = –40.6(c = 0.08, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 7.38–7.28 (m, 5 H), 6.18 (br. d, J = 9.0 Hz,
1 H), 5.33 (br. d, J = 9.0 Hz, 1 H), 5.10 (dd, J = 19.6, 12.0 Hz, 2
H), 4.01 (m, 1 H), 3.92 (m, 1 H), 3.51 (m, 1 H), 1.98–1.77 (m, 2
H), 1.75–1.60 (m, 2 H), 1.39–1.05 (m, 3 H), 1.02–0.83 (m, 18 H)
ppm. 13C NMR (75 MHz, CDCl3): δ = 171.5, 156.4, 136.2, 128.4,
128.0, 127.9, 68.3, 66.9, 60.1, 58.4, 44.1, 36.7, 29.9, 24.5, 24.3, 22.9,
Benzyl [(2S)-1-[(4S)-3-Hydroxy-2,6-dimethylheptan-4-ylamino]-3-
methyl-1-oxobutan-2-yl]carbamate (17c): Compound 17c (106 mg,
53%) was prepared as a colourless oil from the coupling of 12c
(Cbz-l-Val-OH; 189.4 mg, 0.7546 mmol) with the free amine de-
rived from 6 and 6a (80 mg, 0.5031 mmol) following the procedure
described for the synthesis of 16a. Rf = 0.5 (30% EtOAc/hexane).
[α]2D6 = +11.8 (c = 1.2, CHCl3). H NMR (500 MHz, CDCl3): δ =
1
7.39–7.28 (m, 5 H), 6.11 (br. d, J = 8.6 Hz, 1 H), 5.34 (br. d, J =
5.7 Hz, 1 H), 5.11 (ABq, J = 17.2, 12.4 Hz, 2 H), 4.14 (m, 1 H),
3.94 (dd, J = 8.6, 6.7 Hz, 1 H), 3.15 (m, 1 H), 2.31 (br. s, 1 H),
2.13 (m, 1 H), 1.74–1.53 (m, 2 H), 1.48 (m, 1 H), 1.32 (m, 1 H),
1.03–0.85 (m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 170.9,
156.4, 136.1, 128.4, 128.1, 127.9, 78.8, 66.9, 60.7, 48.7, 42.0, 31.1,
22.3, 19.8, 19.3, 15.6, 11.1 ppm. IR (neat): ν = 3421, 2959, 2924,
˜
2854, 1741, 1693, 1516, 1463, 1340, 1315 cm–1. HRMS (ESI): calcd.
for C23H39O4N2 [M + H]+ 407.2909; found 407.2916.
30.8, 24.7, 22.8, 22.4, 19.3, 19.0, 18.9, 17.7 ppm. IR (neat): ν =
˜
3330, 2958, 2924, 2864, 1705, 1649, 1517, 1462, 1391, 1237,
1028 cm–1. HRMS (ESI): calcd. for C22H37O4N2 [M + H]+
393.2753; found 393.2762.
Benzyl [(2S)-1-[(3S)-4-Hydroxy-2,6-dimethylheptan-3-ylamino]-1-
oxo-3-phenylpropan-2-yl]carbamate (16e): Compound 16e (155 mg,
70%) was prepared as a solid from coupling of 12e (Cbz-l-Phe-
OH; 225 mg, 0.7547 mmol) with the free amine derived from 11
(80 mg, 0.503 mmol) following the procedure described for the syn-
thesis of 16a. Rf = 0.4 (30% EtOAc/hexane), m.p. 95–97 °C. [α]2D6
Benzyl [(2S,3S)-1-[(4S)-3-Hydroxy-2,6-dimethylheptan-4-ylamino]-
3-methyl-1-oxopentan-2-yl]carbamate (17d): Compound 17d
(147 mg, 72%) was prepared as a white solid from the coupling
of 12d (Cbz-l-Ileu-OH; 200 mg, 0.7547 mmol) with the free amine
derived from 6 and 6a (80 mg, 0.5031 mmol) following the pro-
cedure described for the synthesis of 16a. Rf = 0.4 (30% EtOAc/
hexane). [α]2D6 = –17.4 (c = 0.66, CHCl3). 1H NMR (300 MHz,
CDCl3): δ = 7.40–7.29 (m, 5 H), 6.10 (br. d, J = 9.0 Hz, 1 H), 5.33
(br. d, J = 9.0 Hz, 1 H), 5.16–5.04 (ABq, J = 17.3, 12.0 Hz, 2 H),
4.13 (m, 1 H), 3.97 (m, 1 H), 3.14 (m, 1 H), 1.86 (m, 1 H), 1.70–
1.64 (m, 2 H), 1.63–1.39 (m, 2 H), 1.31 (m, 1 H), 1.11 (m, 1 H),
1.00–0.83 (m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 171.0,
156.4, 136.2, 136.1, 128.4, 128.0, 127.9, 78.7, 67.0, 66.9, 60.0, 48.7,
42.0, 37.0, 31.1, 24.7, 24.5, 22.8, 22.4, 19.0, 19.0, 15.5, 11.1 ppm.
1
= –8.2 (c = 0.69, CHCl3). H NMR (300 MHz, CDCl3): δ = 7.40–
7.18 (m, 10 H), 6.06 (br. d, J = 9.0 Hz, 1 H), 5.35 (br. s, 1 H), 5.08
(dd, J = 19.6, 12.0 Hz, 2 H), 4.43 (m, 1 H), 3.80 (m, 1 H), 3.46 (m,
1 H), 3.16–3.04 (m, 2 H), 1.85–1.56 (m, 2 H), 1.30–1.01 (m, 2 H),
0.98–0.65 (m, 12 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 171.0,
170.9, 156.0, 155.9, 136.5, 136.3, 129.2, 128.7, 128.6, 128.4, 128.1,
128.0, 127.0, 126.9, 68.8, 68.5, 67.0, 58.7, 58.6, 43.8, 43.7, 38.2,
38.1, 38.0, 29.9, 29.7, 24.4, 24.3, 23.1, 22.1, 22.1, 19.7, 19.0,
18.9 ppm. IR (neat): ν = 3314, 2956, 2927, 1695, 1650, 1531, 1461,
˜
1393, 1256, 1047 cm–1. HRMS (ESI): calcd. for C26H37O4N2 [M +
H]+ 441.2753; found 441.2753.
IR (neat): ν = 3330, 2959, 2926, 2872, 1705, 1676, 1649, 1517, 1462,
˜
Benzyl [2-[(4S)-3-Hydroxy-2,6-dimethylheptan-4-ylamino]-2-oxo-
ethyl]carbamate (17a): Compound 17a (119 mg, 54%) was prepared
as a colourless liquid from the coupling of 12a (Cbz-Gly-OH;
197 mg, 0.9433 mmol) with the free amine derived from 6 and 6a
(TFA, CH2Cl2; 100 mg, 0.6289 mmol) following the procedure de-
scribed for the synthesis of 16a. Rf = 0.4 (60% EtOAc/hexane).
1393, 1239, 1033 cm–1. HRMS (ESI): calcd. for C23H39O4N2 [M +
H]+ 407.2909; found 407.2914.
Benzyl [(2S)-1-[(4S)-3-Hydroxy-2,6-dimethylheptan-4-ylamino]-1-
oxo-3-phenylpropan-2-yl]carbamate (17e): Compound 17e (200 mg,
72%) was prepared as a white solid from the coupling of 12e (Cbz-
l-Phe-OH; 282 mg, 0.9433 mmol) with the free amine derived from
6 and 6a (100 mg, 06289 mmol) following the procedure described
for the synthesis of 16a. Rf = 0.4 (30% EtOAc/hexane), m.p. 103–
105 °C. [α]2D6 = –13 (c = 0.46, CHCl3). 1H NMR (300 MHz,
CDCl3): δ = 7.41–7.15 (m, 10 H), 5.97 (br. d, J = 9.2 Hz, 1 H),
5.36 (br. d, J = 6.7 Hz, 1 H), 5.08 (ABq, J = 16.9, 12.2 Hz, 2 H),
4.39 (m, 1 H), 4.06 (m, 1 H), 3.17–2.95 (m, 3 H), 1.87 (m, 1 H),
1.56–1.33 (m, 2 H), 1.31–1.09 (m, 2 H), 1.02–0.78 (m, 12 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 170.4, 155.8, 136.4, 136.1, 129.2,
128.6, 128.5, 128.1, 127.9, 126.9, 78.7, 67.0, 56.6, 56.5, 48.7, 42.0,
1
[α]2D6 = –9.0 (c = 1.02, CHCl3). H NMR (500 MHz, CDCl3): δ =
7.38–7.28 (m, 5 H), 6.22 (br. s, 1 H), 5.60 (br. s, 1 H), 5.11 (s, 2 H),
4.16 (m, 1 H), 3.96 (m, 1 H), 3.84 (m, 1 H), 3.14 (br. d, J = 7.9 Hz,
1 H), 1.58 (m, 1 H), 1.46 (m, 1 H), 1.29 (m, 2 H), 0.98–0.86 (m, 12
H) ppm. 13C NMR (125 MHz, CDCl3): δ = 168.8, 156.5, 156.4,
136.0, 128.4, 128.1, 128.0, 127.9, 78.9, 67.0, 62.5, 48.8, 44.4, 42.5,
41.9, 30.9, 24.6, 22.9, 22.2, 19.1, 18.4 ppm. IR (neat): ν = 3358,
˜
3333, 2956, 2924, 2862, 1707, 1656, 1518, 1461, 1374, 1248, 1173,
1052 cm–1. HRMS (ESI): calcd. for C19H31O4N2 [M + H]+
351.2283; found 351.2286.
Benzyl [(2S)-1-[(4S)-3-Hydroxy-2,6-dimethylheptan-4-ylamino]-1-
oxopropan-2-yl]carbamate (17b): Compound 17b (157 mg, 68 %)
38.4, 30.6, 24.6, 22.8, 22.3, 19.1, 18.7 ppm. IR (neat): ν = 3303,
2955, 2924, 2862, 1705, 1650, 1531, 1461, 1393, 1251, 1147,
˜
Eur. J. Org. Chem. 2014, 1253–1265
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1261

G. Sudhakar, S. R. Bathula et al.
FULL PAPER
1033 cm–1. HRMS (ESI): calcd. for C26H37O4N2 [M + H]+
CDCl3): δ = 208.5, 171.4, 156.3, 136.2, 128.4, 128.1, 127.9, 67.0,
441.2753; found 441.2756.
62.5, 60.5, 49.8, 31.0, 30.1, 24.3, 22.5, 22.3, 19.9, 19.1, 17.7,
16.6 ppm. IR (neat): ν = 3393, 1741, 1706, 1693, 1516, 1463, 1340,
˜
Benzyl [(2S)-1-[(4S)-3-Hydroxy-2,6-dimethylheptan-4-ylamino]-4-
methyl-1-oxopentan-2-yl]carbamate (17f): Compound 17f (153 mg,
75%) was prepared as a solid from the coupling of 12 (Cbz-l-Leu-
OH; 197.7 mg, 0.7547 mmol) with the free amine derived from 6
and 6a (80 mg, 0.5031 mmol) following the procedure described for
the synthesis of 16a, m.p. 97–99 °C; Rf = 0.4 (30% EtOAc/hexane).
1172 cm–1. HRMS (ESI): calcd. for C22H35O4N2 [M + H]+
391.2596; found 391.2602.
Benzyl [(2S,3S)-1-[(S)-2,6-Dimethyl-4-oxoheptan-3-ylamino]-3-
methyl-1-oxopentan-2-yl]carbamate (18d): Ketone 18d (134 mg,
85 %) was prepared as a white solid from 16d (158 mg,
0.3899 mmol) following the procedure described for the synthesis
1
[α]2D6 = –26.4 (c = 0.51, CHCl3). H NMR (300 MHz, CDCl3): δ =
of 18a. Rf = 0.5 (20% EtOAc/hexane), m.p. 106–108 °C. [α]2D6
=
7.43–7.29 (m, 5 H), 6.33 (br. d, J = 7.5 Hz, 1 H), 5.28 (br. d, J =
8.3 Hz, 1 H), 5.16–5.03 (m, 2 H), 4.24–4.03 (m, 2 H), 3.15 (br. d,
J = 6.0 Hz, 1 H), 1.74–1.41 (m, 6 H), 1.30 (m, 1 H), 1.06–0.82 (m,
18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 171.9, 156.2, 136.1,
128.4, 128.1, 127.9, 78.8, 66.9, 53.7, 48.7, 41.9, 41.4, 31.1, 24.6,
1
+25.8 (c = 0.32, CHCl3). H NMR (500 MHz, CDCl3): δ = 7.37–
7.28 (m, 5 H), 6.38 (br. d, J = 8.7 Hz, 1 H), 5.35 (br. d, J = 7.6 Hz,
1 H), 5.15–5.07 (m, 2 H), 4.58 (dd, J = 8.7, 4.3 Hz, 1 H), 4.07 (m,1
H), 2.45–2.29 (m, 2 H), 2.23–2.13 (m, 2 H), 1.85 (m, 1 H), 1.50 (m,
1 H), 1.15 (m, 1 H), 1.00–0.87 (m, 16 H), 0.81–0.73 (m, 2 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 208.4, 171.3, 156.1, 136.2, 128.4,
128.1, 128.0, 66.9, 62.5, 59.7, 49.8, 37.4, 30.1, 24.7, 24.3, 22.5, 22.3,
22.9, 22.7, 22.4, 22.1, 22.1, 19.0, 18.8 ppm. IR (neat): ν = 3331,
˜
2957, 2926, 2870, 1705, 1694, 1518, 1463, 1394, 1258, 1119,
1046 cm–1. HRMS (ESI): calcd. for C23H39O4N2 [M + H]+
407.2909; found 407.2914.
19.9, 16.7, 15.4, 11.3 ppm. IR (neat): ν = 3301, 2962, 1741, 1693,
˜
1647, 1516, 1463, 1395, 1367 cm–1. HRMS (ESI): calcd. for
C23H37O4N2 [M + H]+ 405.2753; found 405.2759.
(S)-Benzyl [2-(2,6-Dimethyl-4-oxoheptan-3-ylamino)-2-oxoethyl]-
carbamate (18a): DMP (164 mg, 0.387 mmol) was added to a
stirred solution of alcohol 16a (113 mg, 0.3228 mmol) in CH2Cl2
(3 mL) at 0 °C. The resulting solution was stirred at room temp.,
and the reaction was monitored by TLC. Then the reaction mixture
was quenched with a mixture of saturated aq. NaHCO3 and satu-
rated aq. Na2S2O3 (1:1), and the mixture was stirred for a further
15 min to give a clear solution. The aqueous layer was extracted
with EtOAc. The organic layer was washed with brine, dried with
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash column chromatography to give
ketone 18a (95 mg, 84%) as a colourless liquid. Rf = 0.4 (50%
EtOAc/hexane). [α]2D6 = +44.3 (c = 0.49, CHCl3). 1H NMR
(500 MHz, CDCl3): δ = 7.41–7.28 (m, 5 H), 6.57 (br. d, J = 5.5 Hz,
1 H), 5.45 (br. s, 1 H), 5.14 (s, 2 H), 4.61 (dd, J = 8.8, 4.4 Hz, 1
H), 3.98–3.85 (m, 2 H), 2.43–2.31 (d, 2 H), 2.24–2.12 (m, 2 H),
1.01–0.72 (m, 12 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 208.5,
169.1, 156.5, 136.1, 128.3, 127.9, 127.8, 66.9, 62.4, 49.6, 44.3, 29.9,
Benzyl [(S)-1-[(S)-2,6-Dimethyl-4-oxoheptan-3-ylamino]-1-oxo-3-
phenylpropan-2-yl]carbamate (18e): Ketone 18e (142 mg, 92%) was
prepared as a white solid from 16e (155 mg, 0.3521 mmol) follow-
ing the procedure described for the synthesis of 18a. Rf = 0.5 (20%
EtOAc/hexane), m.p. 94–96 °C. [α]2D5.7 = +6.0 (c = 0.33, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 7.46–7.12 (m, 10 H), 6.41 (br. d,
J = 7.3 Hz, 1 H), 5.38 (br. d, 7.5, 1 H), 5.16–5.03 (m, 2 H), 4.56–
4.41 (m, 2 H), 3.07 (d, J = 6.7 Hz, 2 H), 2.39–2.26 (m, 2 H), 2.21–
2.03 (m, 2 H), 1.04–0.64 (m, 12 H) ppm. 13C NMR (75 MHz,
CDCl3): δ = 207.9, 170.8, 155.8, 136.1, 129.2, 128.6, 128.4, 128.1,
128.0, 127.0, 67.0, 62.6, 56.2, 49.7, 38.3, 30.1, 24.2, 22.5, 22.4, 19.8,
16.6 ppm. IR (neat): ν = 3298, 2958, 2926, 1740, 1692, 1650, 1531,
˜
1462, 1395, 1368, 1286, 1039 cm–1. HRMS (ESI): calcd. for
C26H35O4N2 [M + H]+ 439.2596; found 439.2599.
(S)-Benzyl [2-(2,6-Dimethyl-3-oxoheptan-4-ylamino)-2-oxoethyl]-
carbamate (19a): Ketone 19a (98 mg, 83%) was prepared as a yel-
low liquid from 17a (119 mg, 0.340 mmol) following the procedure
described for the synthesis of 18a. Rf = 0.4 (50% EtOAc/hexane).
[α]2D6 = +14.4 (c = 0.38, CHCl3). 1H NMR (300 MHz, CDCl3): δ =
7.39–7.28 (m, 5 H), 6.55 (br. d, J = 8.3 Hz, 1 H), 5.48 (m, 1 H),
5.12 (s, 2 H), 4.83 (m, 1 H), 3.99–3.81 (m, 2 H), 2.79 (m, 1 H),
1.71–1.46 (m, 2 H), 1.35 (m, 1 H), 1.17–1.06 (m, 6 H), 1.00–0.86
(m, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 213.3, 168.8,
156.4, 136.1, 128.4, 128.0, 127.9, 67.0, 54.7, 44.2, 40.4, 37.9, 24.9,
24.0, 22.4, 22.3, 19.7, 16.5 ppm. IR (neat): ν = 3392, 1741, 1693,
˜
1647, 1516, 1463, 1426, 1396 cm–1. HRMS (ESI): calcd. for
C19H29O4N2 [M + H]+ 349.2127; found 349.2131.
Benzyl [(S)-1-[(S)-2,6-Dimethyl-4-oxoheptan-3-ylamino]-1-oxo-
propan-2-yl]carbamate (18b): Ketone 18b (102 mg, 82%) was pre-
pared as a white solid from 16b (125 mg, 0.3434 mmol) following
the procedure described for the synthesis of 18a. Rf = 0.4 (40%
EtOAc/hexane), m.p. 88–90 °C. [α]2D6 = –2.5 (c = 0.2, CHCl3). 1H
NMR (300 MHz, CDCl3): δ = 7.41–7.28 (m, 5 H), 6.63 (br. d, J =
7.5 Hz, 1 H), 5.43 (br. d, J = 6.7 Hz, 1 H), 5.11 (s, 2 H), 4.57 (q, J
= 4.5 Hz, 1 H), 4.31 (m, 1 H), 2.42–2.30 (m, 2 H), 2.27–2.09 (m, 3
H), 1.43–1.32 (m, 3 H), 1.02–0.67 (m, 12 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 208.4, 172.4, 155.8, 136.1, 128.4, 128.1,
127.9, 66.9, 62.5, 50.5, 49.7, 30.1, 30.0, 24.1, 22.5, 22.4, 19.8,
23.2, 21.5, 18.8, 17.5 ppm. IR (neat): ν = 3333, 2960, 2924, 2856,
˜
1708, 1676, 1658, 1517, 1462, 1394, 1242, 1048 cm–1. HRMS (ESI):
calcd. for C19H29O4N2 [M + H]+ 349.2127; found 349.2133.
Benzyl [(S)-1-[(S)-2,6-Dimethyl-3-oxoheptan-4-ylamino]-1-oxo-
propan-2-yl]carbamate (19b): Ketone 19b (141 mg, 90%) was pre-
pared as a yellow liquid from 17b (157 mg, 0.4313 mmol) following
the procedure described for the synthesis of 18a. Rf = 0.4 (40%
EtOAc/hexane). [α]2D5.7 = –11.7 (c = 0.53, CHCl3). 1H NMR
18.5 ppm. IR (neat): ν = 3392, 1741, 1693, 1647, 1516, 1463,
˜
1315 cm–1. HRMS (ESI): calcd. for C20H31O4N2 [M + H]+
363.2283; found 363.2288.
Benzyl [(S)-1-[(S)-2,6-Dimethyl-4-oxoheptan-3-ylamino]-3-methyl-1- (300 MHz, CDCl3): δ = 7.40–7.29 (m, 5 H), 6.50 (br. d, J = 7.5 Hz,
oxobutan-2-yl]carbamate (18c): Ketone 18c (126 mg, 89%) was pre-
pared as a white solid from 16c (126 mg, 0.3230 mmol) following
the procedure described for the synthesis of 18a. Rf = 0.4 (20%
EtOAc/hexanes), m.p. 114–116 °C. [α]2D6 = +12.4 (c = 0.18, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 7.40–7.28 (m, 5 H), 6.45 (br. d,
J = 8.1 Hz, 1 H), 5.39 (br. d, J = 7.5 Hz, 1 H), 5.11 (s, 2 H), 4.59
1 H), 5.37 (br. d, J = 6.0 Hz, 1 H), 5.11 (s, 2 H), 4.79 (m, 1 H),
4.27 (m, 1 H), 2.80 (m, 1 H), 1.68–1.47 (m, 2 H), 1.43–1.30 (m, 4
H), 1.19–1.04 (m, 6 H), 1.00–0.84 (m, 6 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 213.1, 172.1, 155.7, 136.0, 128.3, 127.9,
127.7, 66.6, 54.6, 50.2, 40.1, 37.7, 24.7, 23.1, 21.5, 18.7, 17.5 ppm.
IR (neat): ν = 3314, 2962, 2931, 1707, 1675, 1657, 1517, 1462, 1244,
˜
(dd, J = 8.6, 4.1 Hz, 1 H), 4.05 (m, 1 H), 2.45–2.30 (m, 2 H), 1066, 1038 cm–1. HRMS (ESI): calcd. for C20H31O4N2 [M + H]+
2.26–2.04 (m, 3 H), 1.03–0.69 (m, 18 H) ppm. 13C NMR (75 MHz,
363.2283; found 363.2288.
1262
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 1253–1265

Synthesis and Cytotoxicity of Piperazirum
Benzyl [(S)-1-[(S)-2,6-Dimethyl-3-oxoheptan-4-ylamino]-3-methyl-1- filtered through a pad of Celite, which was then washed with
oxobutan-2-yl]carbamate (19c): Ketone 19c (85 mg, 80%) was pre-
pared as a white solid from 17c (106 mg, 0.2704 mmol) following
the procedure described for the synthesis of 18a. Rf = 0.5 (20%
EtOAc/hexane), m.p. 129–131 °C. [α]2D6 = +3.8 (c = 0.92, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 7.42–7.28 (m, 5 H), 6.44 (br. d,
J = 4.7 Hz, 1 H), 5.42 (br. d, J = 8.4 Hz, 1 H), 5.11 (s, 2 H), 4.83
(m, 1 H), 4.05 (m, 1 H), 2.80 (m, 1 H), 2.12 (m, 1 H), 1.63 (m, 1
EtOAc. The solvent was evaporated under reduced pressure, and
the residue was purified by column chromatography to give cyclic
compound 20a (42 mg, 77 %) as a pale yellow liquid. Rf = 0.2
(EtOAc). [α]2D6 = –9.4 (c = 0.45, MeOH). 1H NMR (500 MHz,
CDCl3): δ = 6.09 (br. s, 1 H), 3.53–3.44 (m, 2 H), 3.22–3.08 (m, 2
H), 1.80–1.66 (m, 2 H), 1.52 (m, 1 H), 1.09 (m, 1 H), 1.02–0.85 (m,
12 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 170.9, 57.2, 51.4,
H), 1.51 (m, 1 H), 1.37 (m, 1 H), 1.19–1.04 (m, 6 H), 1.01–0.85 (m, 47.7, 32.6, 28.7, 23.9, 23.6, 21.1, 19.3, 18.4 ppm. IR (neat): ν =
˜
12 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 213.1, 170.9, 156.2,
128.4, 128.0, 127.9, 66.9, 60.1, 54.6, 40.6, 38.1, 31.3, 24.9, 23.3,
3362, 2960, 2928, 1646, 1367, 1016 cm–1. HRMS (ESI): calcd. for
C11H23ON2 [M + H]+ 199.1810; found 199.1812.
21.6, 19.0, 18.9, 17.7, 17.5 ppm. IR (neat): ν = 3302, 2962, 2928,
˜
(3S,5R,6S)-5-Isobutyl-6-isopropyl-3-methylpiperazin-2-one (20b):
Cyclic compound 20b (43 mg, 72%) was prepared as a pale yellow
solid from 18b (102 g, 0.2817 mmol) following the procedure de-
scribed for the synthesis of 20a. Rf = 0.4 (70% EtOAc/hexane),
m.p. 80–82 °C. [α]2D6 = –29.1 (c = 0.3, MeOH). 1H NMR (300 MHz,
CDCl3): δ = 6.16 (br. s, 1 H), 3.52 (q, J = 6.7 Hz, 1 H), 3.20 (m, 1
H), 3.09 (m, 1 H), 1.93 (m, 1 H), 1.66 (m, 1 H), 1.41–1.21 (m, 5
H), 1.04–0.85 (m, 12 H) ppm. 13C NMR (75 MHz, CDCl3): δ =
174.0, 59.6, 54.5, 53.3, 40.6, 27.8, 24.7, 23.1, 22.4, 21.5, 18.2,
2874, 1707, 1650, 1531, 1462, 1388, 1374, 1287, 1238, 1100,
1035 cm–1. HRMS (ESI): calcd. for C22H29O4N2 [M + H]+
391.2596; found 391.2601.
Benzyl [(2S,3S)-1-[(S)-2,6-Dimethyl-3-oxoheptan-4-ylamino]-3-
methyl-1-oxopentan-2-yl]carbamate (19d): Ketone 19d (129 mg,
88%) was prepared as a white solid from 17d (147 mg, 0.362 mmol)
following the procedure described for the synthesis of 18a. Rf = 0.4
(20% EtOAc/hexane), m.p. 119–121 °C. [α]2D6 = –2.3 (c = 0.53,
CHCl3). 1H NMR (300 MHz, CDCl3): δ = 7.39–7.29 (m, 5 H), 6.29
(br. d, J = 7.5 Hz, 1 H), 5.35 (br. d, J = 8.3 Hz, 1 H), 5.10 (s, 2 H),
4.81 (m, 1 H), 4.04 (m, 1 H), 2.79 (m, 1 H), 1.83 (m, 1 H), 1.71–
1.57 (m, 2 H), 1.56–1.43 (m, 2 H), 1.36 (m, 1 H), 1.17–1.06 (m, 6
H), 0.99–0.83 (m, 12 H) ppm. 13C NMR (75 MHz, CDCl3): δ =
213.0, 170.9, 156.1, 136.2, 128.4, 128.0, 127.9, 66.9, 59.5, 54.6, 40.6,
38.1, 37.6, 24.9, 24.7, 23.3, 21.6, 18.8, 17.5, 15.3, 11.3 ppm. IR
17.6 ppm. IR (neat): ν = 3278, 2959, 2929, 1648, 1369 cm–1. HRMS
˜
(ESI): calcd. for C12H23ON2 [M – H]+ 211.1810; found 211.1812.
(3S,5R,6S)-5-Isobutyl-3,6-diisopropylpiperazin-2-one (20c): Cyclic
compound 20c (50 mg, 64%) was prepared as a liquid from 18c
(126 mg, 0.3230 mmol) following the procedure described for the
synthesis of 20a. Rf = 0.4 (30% EtOAc/hexane). [α]2D6 = –44.7 (c =
1
0.24, MeOH). H NMR (500 MHz, CDCl3): δ = 5.61 (br. s, 1 H),
(neat): ν = 3283, 2962, 2924, 1741, 1693, 1646, 1516, 1463, 1395,
˜
3.25 (d, J = 2.7 Hz, 1 H), 2.67 (dt, J = 9.1, 2.7 Hz, 1 H), 2.48 (m,
1 H), 1.93 (m, 1 H), 1.79 (m, 1 H), 1.37–1.22 (m, 3 H), 1.07–0.81
(m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 173.3, 63.6, 63.3,
52.7, 40.9, 29.1, 27.6, 24.5, 23.8, 21.3, 19.8, 19.3, 16.5, 14.3 ppm.
1367, 1037 cm–1. HRMS (ESI): calcd. for C23H37O4N2 [M + H]+
405.2753; found 405.2757.
Benzyl [(S)-1-[(S)-2,6-Dimethyl-3-oxoheptan-4-ylamino]-1-oxo-3-
phenylpropan-2-yl]carbamate (19e): Ketone 19e (188 mg, 94%) was
prepared as a white solid from 17e (200 mg, 0.4545 mmol) follow-
ing the procedure described for the synthesis of 18a. Rf = 0.4 (20%
EtOAc/hexanes), m.p. 119–121 °C. [α]2D6 = –5.7 (c = 0.35, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 7.42–7.12 (m, 10 H), 6.25 (br. d,
J = 8.3 Hz, 1 H), 5.31 (br. d, J = 7.5 Hz, 1 H), 5.15–5.05 (m, 2 H),
4.74 (m, 1 H), 4.44 (m, 1 H), 3.17–2.98 (m, 2 H), 2.72 (m, 1 H),
1.57–1.18 (m, 3 H), 1.15–1.03 (m, 6 H), 0.97–0.82 (m, 6 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 212.4, 170.3, 155.7, 136.1, 129.2,
128.6, 128.5, 128.1, 127.9, 127.0, 67.0, 56.0, 54.6, 40.8, 38.4, 37.9,
IR (neat): ν = 3209, 2959, 2925, 2863, 1659, 1463, 1339 cm–1
.
˜
HRMS (ESI): calcd. for C14H29ON2 [M + H]+ 241.2279; found
241.2278.
(3S,5R,6S)-3-sec-butyl-5-isobutyl-6-isopropylpiperazin-2-one (20d):
Cyclic compound 20d (48 mg, 56%) was prepared as a liquid from
18d (134 mg, 0.3353 mmol) following the procedure described for
the synthesis of 20a. Rf = 0.35 (30% EtOAc/hexane). [α]2D6 = –31.8
1
(c = 0.4, MeOH). H NMR (500 MHz, CDCl3): δ = 5.58 (br. s, 1
H), 3.28 (d, J = 2.0 Hz, 1 H), 3.00 (dd, J = 8.9, 2.0 Hz, 1 H), 2.65
(dt, J = 9.9, 2.9 Hz, 1 H), 2.18 (m, 1 H), 1.93 (m, 1 H), 1.80 (m, 1
H), 1.44 (m, 1 H), 1.36–1.21 (m, 3 H), 1.04–0.82 (m, 18 H) ppm.
13C NMR (125 MHz, CDCl3): δ = 173.2, 63.9, 63.4, 52.9, 40.9,
36.1, 27.6, 24.5, 24.4, 23.9, 21.3, 19.8, 16.1, 14.3, 12.3 ppm. IR
24.8, 23.2, 21.7, 18.9, 17.5 ppm. IR (neat): ν = 3280, 2961, 2924,
˜
1741, 1647, 1517, 1463, 1426, 1395, 1340, 1263, 1051 cm–1. HRMS
(ESI): calcd. for C26H35O4N2 [M + H]+ 439.2596; found 439.2599.
(neat): ν = 3234, 2962, 2874, 1648, 1368 cm–1. HRMS (ESI): calcd.
˜
Benzyl [(S)-1-[(S)-2,6-Dimethyl-3-oxoheptan-4-ylamino]-4-methyl-1-
oxopentan-2-yl]carbamate (19f): Ketone 19f (131 mg, 86%) was pre-
pared as a white solid from 17f (153 mg, 0.3768 mmol) following
the procedure described for the synthesis of 18a. Rf = 0.4 (20%
EtOAc/hexanes), m.p. 125–127 °C. [α]2D6 = –7.9 (c = 0.6, CHCl3).
1H NMR (300 MHz, CDCl3): δ = 7.40–7.29 (m, 5 H), 6.39 (br. d,
J = 8.3 Hz, 1 H), 5.22–5.08 (m, 3 H), 4.79 (m, 1 H), 4.20 (m, 1 H),
2.80 (m, 1 H), 1.72–1.42 (m, 6 H), 1.18–1.06 (m, 6 H), 1.00–0.86
(m, 12 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 213.0, 171.9,
156.0, 136.1, 128.4, 128.0, 127.9, 66.9, 54.7, 53.4, 41.5, 40.5, 37.9,
for C15H29ON2 [M – H]+ 253.2279; found 253.2282.
(3S,5R,6S)-3-Benzyl-5-isobutyl-6-isopropylpiperazin-2-one (20e):
Cyclic compound 20e (75 mg, 80%) was prepared as a liquid from
18e (142 mg, 0.3239 mmol) following the procedure described for
the synthesis of 20a. Rf = 0.4 (50% EtOAc/hexane). [α]2D6 = –59.4
(c = 0.29, MeOH). 1H NMR (300 MHz, CDCl3): δ = 7.35–7.17 (m,
5 H), 6.41 (br. s, 1 H), 3.64 (dd, J = 8.3, 3.7 Hz, 1 H), 3.33 (dd, J
= 13.5, 3.0 Hz, 2 H), 3.16–3.01 (m, 2 H), 2.93 (dd, J = 13.5, 8.3 Hz,
1 H), 1.83 (m, 1 H), 1.50 (m, 1 H), 1.35–1.14 (m, 2 H), 0.98–0.69
(m, 12 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 172.7, 138.1,
129.5, 128.3, 126.4, 59.9, 59.0, 53.3, 40.6, 37.8, 27.6, 24.6, 22.6,
24.9, 24.5, 23.3, 22.8, 21.9, 21.6, 18.9, 17.5 ppm. IR (neat): ν =
˜
3297, 2959, 2928, 2868, 1694, 1647, 1545, 1461, 1266, 1248, 1126,
1048 cm–1. HRMS (ESI): calcd. for C23H37O4N2 [M + H]+
405.2753; found 405.2756.
22.6, 21.5, 17.1 ppm. IR (neat): ν = 3217, 2957, 2927, 2870, 1659,
˜
1562, 1516, 1476, 1426, 1276 cm–1. HRMS (ESI): calcd. for
C18H29ON2 [M + H]+ 289.2274; found 289.2274.
(5R,6S)-5-Isobutyl-6-isopropylpiperazin-2-one (20a): A solution of
ketone 18a (95 mg, 0.2743 mmol) in MeOH (3 mL) was hydroge-
nated at balloon pressure (1 atm) in the presence of 10% Pd/C
(95 mg). The reaction was monitored by TLC. The mixture was
(5R,6S)-6-Isobutyl-5-isopropylpiperazin-2-one (21a): Cyclic com-
pound 21a (45 mg, 80%) was prepared as a colourless liquid from
19a (98 mg, 0.2822 mmol) following the procedure described for
Eur. J. Org. Chem. 2014, 1253–1265
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1263

G. Sudhakar, S. R. Bathula et al.
FULL PAPER
the synthesis of 20a. Rf = 0.2 (EtOAc). [α]2D6 = –45.8 (c = 0.12,
from 19f (131 g, 0.3242 mmol) following the procedure described
1
MeOH). H NMR (300 MHz, CDCl3): δ = 6.13 (br. s, 1 H), 3.54 for the synthesis of 20a. Rf = 0.35 (30% EtOAc/hexane). [α]2D6
=
(br. d, J = 6.0 Hz, 2 H), 3.43 (m, 1 H), 2.55 (dd, J = 9.8, 3.3 Hz,
1 H), 1.72 (m, 1 H), 1.63–1.50 (m, 2 H), 1.13 (m, 1 H), 1.04 (d, J
= 6.4 Hz, 3 H), 0.98 (d, J = 6.4 Hz, 3 H), 0.91 (d, J = 6.6 Hz, 6 H)
ppm. 13C NMR (75 MHz, CDCl3): δ = 170.5, 62.4, 51.1, 49.9, 38.0,
–22.5 (c = 0.2, MeOH). 1H NMR (300 MHz, CDCl3): δ = 5.86 (br.
s, 1 H), 3.39–3.25 (m, 2 H), 2.40 (dd, J = 9.6, 2.4 Hz, 1 H), 1.92
(m, 1 H), 1.85–1.60 (m, 2 H), 1.47 (m, 1 H), 1.35–1.21 (m, 3 H),
1.08–0.82 (m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ = 173.5,
28.6, 24.2, 23.7, 20.8, 20.3, 19.2 ppm. IR (neat): ν = 3220, 2958, 62.6, 57.2, 53.7, 42.1, 40.3, 27.3, 24.6, 23.9, 23.9, 23.5, 21.0, 21.0,
˜
2928, 2874, 1692, 1248 cm–1. HRMS (ESI): calcd. for C11H23ON2 20.6, 15.0 ppm. IR (neat): ν = 3203, 2956, 2926, 2869, 1660, 1309,
˜
[M + H]+ 199.1810; found 199.1811.
1159 cm–1. HRMS (ESI): calcd. for C15H31ON2 [M + H]+
255.2436; found 255.2434.
(3S,5R,6S)-6-Isobutyl-5-isopropyl-3-methylpiperazin-2-one (21b):
Cyclic compound 21b (65 mg, 78%) was prepared as a colourless
liquid from 19b (141 mg, 0.3895 mmol) following the procedure de-
scribed for the synthesis of 20a. Rf = 0.4 (70% EtOAc/hexane).
[α]2D6 = –86.3 (c = 0.11, MeOH). 1H NMR (300 MHz, CDCl3): δ =
6.39 (br. s, 1 H), 3.52 (q, J = 6.7 Hz, 1 H), 3.38 (m, 1 H), 2.62 (dd,
J = 9.8, 3.0 Hz, 1 H), 1.79–1.50 (m, 3 H), 1.36 (d, J = 6.7 Hz, 3
H), 1.13 (m, 1 H), 1.03 (d, J = 6.0 Hz, 3 H), 0.96 (d, J = 6.7 Hz, 3
H), 0.89 (d, J = 6.0 Hz, 6 H) ppm. 13C NMR (75 MHz, CDCl3): δ
= 173.5, 62.5, 55.0, 51.5, 38.5, 28.4, 24.2, 23.8, 20.8, 20.2, 19.0,
Biological Evaluation
Materials and Methods: Cancer cell lines (A549, SK-OV-3, DU-
145, MDA-MB-231, MCF-7) and non-cancer cell lines (HEK-293
and NIH/3T3) used in the evaluation were acquired from ATCC
(American Type Cell Culture). 3-(4,5-Dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) dye and Annexin V were pur-
chased from Sigma–Aldrich. An ELISA (enzyme-linked immuno-
sorbent assay) plate reader was used to record the absorbance in
the MTT assay.
18.4 ppm. IR (neat): ν = 3277, 2964, 2931, 2875, 1650, 1236 cm–1.
˜
HRMS (ESI): calcd. for C12H25N2O [M + H]+ 213.1967; found
213.1972.
Cell Culture: Cells were kept in RPMI (Roswell Park Memorial
Institute medium) 1640 constituting Fetal Bovine Serum (10%) and
antibiotic and antimycotic solution (1%) under optimal growth
conditions (temperature: 37 °C, humidity: 95%, and CO2: 5%). The
stock solution was prepared in molecular grade DMSO. The de-
sired concentrations (2.5, 5, 10, 20, 50, and 100 μm) of the com-
pounds were obtained by diluting the stock solution with culture
media before addition to the cells.
(3S,5R,6S)-6-Isobutyl-3,5-diisopropylpiperazin-2-one (21c): Cyclic
compound 21c (45 mg, 86%) was prepared as a colourless liquid
from 19c (85 mg, 0.2179 mmol) following the procedure described
for the synthesis of 20a. Rf = 0.35 (40% EtOAc/hexane). [α]2D6
=
–64.7 (c = 0.49, MeOH). 1H NMR (500 MHz, CDCl3): δ = 6.37
(br. s, 1 H), 3.38 (d, J = 2.2 Hz, 1 H), 2.56 (dd, J = 9.9, 3.3 Hz, 1
H), 2.44 (m, 1 H), 1.76–1.65 (m, 3 H), 1.59 (m, 1 H), 1.11 (m, 1
H), 1.05–0.85 (m, 18 H) ppm. 13C NMR (75 MHz, CDCl3): δ =
172.9, 64.0, 62.1, 51.1, 38.4, 29.8, 28.4, 24.3, 23.7, 20.9, 20.0, 19.0,
In-vitro Cytotoxicity Assay: The compound corresponding to the
proposed structure of piperazirum was tested, along with its stereo-
isomers and analogues, for in vitro anticancer activity across dif-
ferent cancer cell lines (A549, SK-OV-3, DU145, MDA-MB-231,
MCF-7) and non-cancer cell lines (HEK-293 and NIH/3T3) using
a colorimetric MTT assay. Briefly, cells were incubated in 96-well
plates (4ϫ103 cells/well) for 24 h. After 24 h, cells were treated with
the test compound at concentrations of 2.5, 5, 10, 20, 50, and
100 μm, and the resulting mixtures were incubated for 48 h. At the
end of the incubation period, MTT (10 mg/mL; 10 μL) was added
to each well, and the mixtures were incubated for a further 3 h.
The intensity of purple coloured water-insoluble formazan dye [sol-
ubilized by the addition of DMSO (100 μL) to each well] formed
due to the reduction of MTT by living cells was recorded using an
ELISA plate reader at 540 nm. All the experiments were performed
at least three times independently, and IC50 values were calculated
using Graph pad prism software.
18.9, 16.4 ppm. IR (neat): ν = 3198, 2956, 1661, 1159, 1079 cm–1.
˜
HRMS (ESI): calcd. for C14H29ON2 [M + H]+ 241.2274; found
241.2272.
(3S,5R,6S)-3-sec-Butyl-6-isobutyl-5-isopropylpiperazin-2-one (21d):
Cyclic compound 21d (50 mg, 62%) was prepared as a liquid from
19d (129 mg, 0.319 mmol) following the procedure described for
the synthesis of 20a. Rf = 0.4 (30% EtOAc/hexane). [α]2D6 = –45.0
1
(c = 0.4, MeOH). H NMR (500 MHz, CDCl3): δ = 6.37 (br. s, 1
H), 3.39 (s, 1 H), 3.34 (m, 1 H), 2.54 (dd, J = 10.4, 2.7 Hz, 1 H),
2.12 (br. s, 1 H), 1.74–1.63 (m, 2 H), 1.57 (m, 1 H), 1.41 (m, 1 H),
1.32 (m, 1 H), 1.10 (m, 1 H), 1.03–0.98 (m, 6 H), 0.97–0.93 (m, 3
H), 0.92–0.86 (m, 9 H) ppm. 13C NMR (75 MHz, CDCl3): δ =
173.3, 64.3, 62.3, 51.0, 38.4, 36.8, 28.3, 24.2, 23.7, 20.9, 20.0, 18.9,
15.7, 12.3 ppm. IR (neat): ν = 3206, 2958, 2852, 1657, 1395,
˜
1367 cm–1. HRMS (ESI): calcd. for C15H31N2O [M + H]+
255.2430; found 255.2428.
Supporting Information (see footnote on the first page of this arti-
cle): Copies of the 1H and 13C NMR spectra of all new compounds,
and crystallographic data for compound 15.
(3S,5R,6S)-3-Benzyl-6-isobutyl-5-isopropylpiperazin-2-one (21e):
Cyclic compound 21e (94 mg, 76%) was prepared as a colourless
liquid from 19e (188 mg, 0.4292 mmol) following the procedure de-
scribed for the synthesis of 20a. Rf = 0.35 (50% EtOAc/hexane).
[α]2D6 = –33.3 (c = 0.15, MeOH). 1H NMR (300 MHz, CDCl3): δ =
7.35–7.20 (m, 5 H), 6.54 (br. s, 1 H), 3.69 (t, J = 6.0 Hz, 1 H), 3.29
(m, 1 H), 3.15 (d, J = 6.0 Hz, 2 H), 2.53 (dd, J = 9.8, 3.0 Hz, 1 H),
1.59–1.33 (m, 2 H), 0.99–0.87 (m, 4 H), 0.85–0.75 (m, 10 H) ppm.
13C NMR (75 MHz, CDCl3): δ = 172.5, 137.7, 129.6, 128.3, 126.5,
61.9, 59.7, 51.2, 38.0, 37.4, 28.5, 24.0, 23.6, 20.7, 20.1, 19.0 ppm.
Acknowledgments
The authors thank the Council of Scientific and Industrial Re-
search (CSIR), New Delhi, India for financial support as part of
the XII Five-Year plan programme under the titles ORIGIN (CSC-
0108) and ADD (CSC-0302). The CSIR and the University Grants
Commission (UGC), New Delhi, are thanked for the award of re-
search fellowships (S. B. and K. J. R.). K. S. is grateful to the De-
partment of Science and Technology (DST) for an INSPIRE fel-
lowship.
IR (neat): ν = 3305, 1741, 1693, 1647, 1516, 1463, 1426, 1395, 1367,
˜
1315 cm–1. HRMS (ESI): calcd. for C18H29ON2 [M + H]+
289.2279; found 289.2281.
(3S,5R,6S)-3,6-Diisobutyl-5-isopropylpiperazin-2-one (21f): Cyclic
compound 21f (68 mg, 82%) was prepared as a colourless liquid
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Eur. J. Org. Chem. 2014, 1253–1265

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Received: September 19, 2013
Published Online: December 12, 2013
Eur. J. Org. Chem. 2014, 1253–1265
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1265