Synthesis and biological activity of N-arylpiperazine-modified analogues of KN-62, a potent antagonist of the purinergic P2X7 receptor

Article abstract of DOI:10.1021/jm021049d

The P2X₇ receptor is involved in several processes relevant to inflammation (cytokine release, NO generation, killing of intracellular pathogens, cytotoxicity); thus, it may be an appealing target for pharmacological intervention. The character

Full text of DOI:10.1021/jm021049d

1318
J . Med. Chem. 2003, 46, 1318-1329
Syn th esis a n d Biologica l Activity of N-Ar ylp ip er a zin e-Mod ified An a logu es of
KN-62, a P oten t An ta gon ist of th e P u r in er gic P 2X7 Recep tor
Pier Giovanni Baraldi,*,†,# Maria del Carmen Nun˜ez,† Anna Morelli,‡ Simonetta Falzoni,‡
Francesco Di Virgilio,‡,# and Romeo Romagnoli†
Dipartimento di Scienze Farmaceutiche, Dipartimento di Medicina Diagnostica e Sperimentale, Sezione di Patologia Generale,
and Interdisciplinary Center for the Study of Inflammation (ICSI), Universita` di Ferrara, 44100 Ferrara, Italy
Received September 26, 2002
The P2X7 receptor is involved in several processes relevant to inflammation (cytokine release,
NO generation, killing of intracellular pathogens, cytotoxicity); thus, it may be an appealing
target for pharmacological intervention. The characterization of native and recombinant P2X7
receptor continues to be hindered by the lack of specific and subtype-selective antagonists.
However, a tyrosine derivative named KN-62 exhibits selective P2X7 receptor-blocking
properties. The present study was designed to evaluate the functional antagonistic properties
of a novel series of KN-62-related compounds characterized by the presence of different phenyl-
substituted piperazine moieties. Antagonistic activity of KN-62 derivatives was tested on
HEK293 cells transduced with the human P2X7 receptor and monocyte-derived human
macrophages, a cell type well-known for the high level of expression of this receptor. The
biological responses investigated were ATP-dependent Ca2+ influx across the plasma membrane,
ethidium bromide uptake, and secretion of the cytokine interleukin-1â. KN-62 was characterized
by the presence of a phenylpiperazine moiety, and the presence of a one-carbon linker between
the piperazine nitrogen and the phenyl ring (compound 61) increases the activity, while a two-
carbon linker (compound 62) decreases biological activity 10-fold. Also, the nature and the
position of substituents on the phenyl ring tethered to the piperazine seemed to exert a
fundamental influence on the biological activity. In the series of synthesized compounds, the
presence of a fluorine in the para position gives the most potent compound (63), while the
same atom in the ortho position reduces potency by 3-fold. When the p-fluorine was replaced
in the same position with other halogens, such as chlorine (compound 64) or iodine (compound
65), the activity decreased dramatically. We then tested the activity of the four most potent
KN-62 derivatives on ATP-stimulated secretion of IL-1â from monocyte-derived human
macrophages, a key cell type in inflammation and innate immunity. Interestingly, compound
68 and 71 caused a complete inhibition of IL-1â release, while with KN-62, 63, and 85, there
was a small residual cytokine secretion even at concentrations exceeding 100 nM. None of the
compounds tested on IL-1â release had any effect on isolated CaMII kinase activity up to 20
µM (not shown).
In tr od u ction
reproductive system (P2X7), motor control (P2X2), in-
flammation and immunity (P2X7), apoptosis (P2X1 and
P2X7), and cardiovascular pathology (P2X4).6,8-10 Un-
fortunately, characterization of native and recombinant
P2 receptor continues to be hindered by the lack of
specific and subtype-selective antagonists.
It has been increasingly recognized that extracellular
adenosine 5′-triphosphate (ATP) acts as an extracellular
messenger by stimulating purinergic P2 receptors,
which are considered a promising new target for anti-
inflammatory drug development.1-4 P2 receptors are
classified into two families comprising seven subtypes
each: G-protein-coupled receptors named P2Y (P2Y1,
P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13) and ligand-
gated ion channels named P2X (P2X1-7).5-7 Both fami-
lies are the focus of great attention because of their
therapeutic potential. In particular, P2X receptors are
under scrutiny as potential targets for the alleviation
of pain (P2X3), modulation of endocrine function and
fertility (P2X4 and P2X7), treatment of cancer of the
The P2X7 receptor differs strikingly from the other
members of the P2X family for its size, 595 amino acids,
for the long (242 residues) C-terminal chain, and for its
peculiar ability to form a nonselective plasma mem-
brane pore of 3-5 nm size when exposed to sustained
stimulation with ATP.11-13 Opening of the P2X7 pore
allows plasma membrane fluxes of ions as well as of
hydrophilic molecules with molecular mass up to 900
Da.11,14-16 The physiological function of P2X7 is as yet
unknown; however, its high expression in immune cells,
including microglia, macrophages, lymphocytes, and
dendritic cells, where it mediates cytotoxic responses,
cytochine release, and cell fusion, suggests that it may
have an important role in immunomodulation.17-19 In
vivo and in vitro studies show that stimulation of the
P2X7 receptor in macrophages and microglial cells is one
* To whom correspondence should be addressed. Address: Dipar-
timento di Scienze Farmaceutiche, Universita` di Ferrara, Via Fossato
di Mortara 17-19, 44100 Ferrara, Italy. Phone: +39-(0)532-291293.
Fax: +39-(0)532-291296. E-mail: pgb@ifeuniv.unife.it.
† Dipartimento di Scienze Farmaceutiche.
#
Dipartimento di Medicina Diagnostica e Sperimentale, Sezione di
Patologia Generale.
‡ Interdisciplinary Center for the Study of Inflammation.
10.1021/jm021049d CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/07/2003

Analogues of KN-62
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1319
of the most potent stimuli for interleukin-1â (IL-1â)
secretion in response to lipopolysaccharide (LPS) stimu-
lation.20,21 IL-1â is of prime importance in the induction
of the immune responses, including facilitating re-
sponses to antigens and synthesis of prostaglandins, and
in the pathogenesis of local and systemic inflammatory
reaction (e.g., septic shock).
Human macrophages have proven to be very useful
for the evaluation of P2X7 agonists and antagonists, and
among the latter, the KN-62 (1-(N,O-bis(1,5-isoquino-
linesulfonyl)-N-methyl-L-tyrosyl)-4-phenylpiperazine)
compound (1) is, to the best of our knowledge, the most
potent antagonist for the human P2X7 receptors with
an IC50 of 51 nM and complete inhibition at 500 nM.22-24
pletely lost the antagonist properties with respect to the
parent compound, and this confirms the notion than an
extended rather than folded conformation of tyrosine
is preferred at the P2X7 receptor.
In the structure of KN-62, the importance of the
presence of the isoquinoline-5-sulfonyl moieties, linked
to the nitrogen and to the hydroxyl group of the
N-methyltyrosine, for binding capacity to the P2X7
receptor has been studied by the synthesis of a series
of KN-62-analogues (compounds 3-6) that possess
different arylsulfonyl moieties. In these latter deriva-
tives, the isoquinoline-5-sulfonyl function that charac-
terizes KN-62 has been substituted, with its two isomers
corresponding to quinoline-5- and quinoline-8-sulfonyl
moieties (compounds 3 and 4, respectively), with the
naphthalene-1-sulfonyl group (compound 5), and finally
with a 3-pyridinesulfonyl moiety (compound 6) (unpub-
lished results). Unfortunately, neither the compounds
3-5 nor the derivative 6 are able to maintain the same
antagonist activity as the parent compound KN-62, and
this means that the isoquinoline-5-sulfonyl moiety is
essential for the activity. In addition, on the lead
compound KN-62, the presence of the methyl on the
R-nitrogen is not essential for the activity. In fact, its
removal is well tolerated without loss of antagonism.27
In a study in which three positions of KN-62 have been
systematically modified corresponding to the R-nitrogen
and the hydroxyl function of the tyrosine moiety, along
with the phenylpiperazine group, J acobson et al. have
identified a compound named MRS 2306 (1-(N,O-bis-
(1,5-isoquinolinesulfonyl)-N-L-tyrosyl)-4-tert-butyloxy-
carbonylpiperazine) that was slightly more potent than
the reference compound KN-62.27
In the present article, we have focused our attention
on the modification of the phenylpiperazine residue. We
have performed a systematic structure-activity analysis
by examining the effects due to the insertion of different
chemical functionalities on the phenyl ring linked to the
piperazine nitrogen. These modifications will alter the
electronic, steric, and lipophilic/hydrophilic features of
the aromatic portion joined to the piperazine, and we
hope to obtain compounds more potent than KN-62.
Ch em istr y
Compounds 61-87 were prepared using (L)-N-Boc-
N-methyltyrosine (7) and (L)-N--Boc-tyrosine (8) as
starting materials,28 following the synthetic sequence
highlighted in Scheme 1. These amino-protected precur-
sors 7 and 8 were converted to the activated 1-hydroxy-
1,2,3-benzotriazole (HOBt) esters with HOBt and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC) and were coupled with the appropriate N-
arylpiperazines29 to give the amides 9-32 and 33, 34,
respectively, in good yields. Compounds 35-58 and 59,
60 were prepared from the corresponding tyrosylphenol
anion of 9-32 and 33, 34, respectively (generated in situ
with sodium hydride), by treatment with a dichloro-
methane (CH2Cl2) solution of 5-isoquinolinesulfonyl
chloride.30,31 The protecting tert-butyloxycarbonyl (Boc)
group in 35-60 was conveniently removed by the use
of trifluoroacetic acid (TFA) in a CH2Cl2 solution and
furnished the corresponding free amine that was then
coupled with an excess of 5-isoquinolinesulfonyl chlo-
ride, providing the sulfonamides 61-86 in acceptable
Recently, our group has studied the effect of confor-
mational restriction of KN-62, by the synthesis of
conformationally constrained KN-62 analogue, with
formula 2, where the tyrosine backbone was replaced
with the 1,2,3,4-tetrahydroisoquinoline (TIC) moiety
that can be considered as a “cyclic tyrosine”.25,26 In this
way, a six-member ring was introduced to freeze the
rotation of the CR-Câ bond of the N-methyltyrosine.
This ring-constrained N-methyltyrosine moiety (TIC)
has been inserted in order to restrict conformational
freedom and to stabilize the desired bioactive conforma-
tion of KN-62 and in an effort to examine the tolerability
of the P2X7 receptor to a rigid tyrosine replacement.
Unfortunately, this constrained form of KN-62 com-

1320 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Baraldi et al.
Sch em e 1a
F igu r e 1. ATP-dependent cytoplasmic Ca2+ increases in wild-
type and P2X7-transduced HEK293 cells. Cells were resus-
pended in the fluorometer cuvette in the saline solution
described in Materials and Methods at a concentration of 106
cells/mL. ATP was 1 mM, and ionomycin was 100 nM. The
P2X7 blocker, compound 71, was added at a concentration of
50 nM 5 min prior to ATP. Black trace, HEK293-P2X7; red
trace, HEK293-P2X7 treated with compound 71 prior to ATP;
green trace, wild-type HEK293.
marized in Table 2. Compound 63 was the most active
antagonist in this series, and several compounds (i.e.,
61, 66, 68, 71, 73-76, 78, 79, 81, 85, and 86) showed
an antagonistic activity more potent than KN-62.
With the aim of investigating the importance of the
distance between the piperazine basic nitrogen and the
phenyl ring by the insertion of one and two methylene
units, compounds 61 and 62 were synthesized and
tested. For compound 61, a methylene spacer slightly
improves the activity, which was 2-fold higher than that
of the reference compound KN-62, while for derivative
62 (one carbon longer than 61) the presence of an ethyl
spacer was detrimental to the antagonist potency, which
was 10- and 30-fold lower than that reported for KN-
62 and 61, respectively. These data suggest that the
optimal length of the linker contained one carbon atom,
which is presumably important for the relative position
of the phenyl ring in the space.
The presence of a basic nitrogen in the piperazine
moiety was important for the activity. In fact, its
removal by the substitution of the piperazine with a
piperidine (compound 84, the 4-benzylpiperidine ana-
logue of 61) reduced the activity by 3-fold (IC50 ) 21.1
vs 65.3 nM for 61 vs 84, respectively).
Results shown in the Table 1 indicate that the
substitution of the phenyl ring with other six-member
ring heterocycles with one nitrogen (pyridine, 82) or two
nitrogens (pyrimidine, 83) results in a 1.5- to 3-fold
reduction in activity, respectively.
a
Reagents: (a) substituted aryl- or heteroarylpiperazine, HOBt
(1.1 equiv), EDC (1.1 equiv), DMF, room temp, 24 h; (b) NaH,
isoquinolinesulfonyle chloride, CH2Cl2, room temp, 18 h; (c) TFA,
CH2Cl2, room temp; (d) TEA, isoquinolinesulfonyle chloride,
CH2Cl2, room temp, 18 h; (e) H2, 10% Pd/C, MeOH, 4 h.
yields. Compound 87 was obtained by a reductive
hydrogenation of 68 in the presence of palladium on
charcoal.
The structures of the synthesized compounds 61-87
and the yields of the syntheses are presented in Table
1. The range of yields was 10-88%.
Resu lts a n d Discu ssion
The antagonist activity of KN-62 derivatives 61-87
modified on the arylpiperazine pharmacophore was
investigated by the measurements of two ATP-depend-
ent responses: ATP-stimulated Ca2+ influx into and
ethidium bromide uptake by HEK293 cells transfected
with the human P2X7 receptor (HEK293-P2X7). Since
HEK293 cells lack endogenous P2X receptors, they are
an ideal cell model for the expression of recombinant
P2X7 and for antagonist potency studies. Figure 1 shows
a typical experiment in which changes in the intra-
cellular Ca2+ concentration as a consequence of P2X7
receptor activation were recorded in wild-type and P2X7-
transduced HEK293 cells.
The effect of one of the KN-62 derivatives synthesized
(see below) is also shown. As expected for a true Ca2+
influx, the P2X7-dependent increase in cytoplasmic Ca2+
was fully abolished in the absence of extracellular Ca2+
(not shown). Results of experiments similar to that
shown in Figure 1 performed with a large number of
compounds synthesized in our laboratory are sum-
For the compounds that possess different substituents
on the phenyl ring in the para position, they showed
variable potencies, where derivative 63, characterized
by the presence of a fluorine, was the most potent
compounds of the whole series, being 30-fold more
potent than KN-62. The other para-halogenated ana-
logues, such as p-chloro (64) and p-iodo (65), showed
lower potencies (from 2- to 3-fold) varying with the

Analogues of KN-62
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1321
Ta ble 1. Physical and Synthetic Data of Novel KN-62 Analogues (61-87)
compd
R1
R2
X
mp, °C
yield,a
%
formulab
anal.
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH2C6H5
N
92-94
35
25
38
61
44
58
47
76
58
43
10
18
40
76
58
58
10
67
20
21
64
48
32
35
50
88
45
C
C
C
C
C
C
C
C
C
C
C
39H37N5O6S2
40H39N5O6S2
38H34N5O6S2F
38H34N5O6S2Cl
38H34N5O6S2I
39H37N5O6S2
39H37N5O7S2
38H34N6O8S2
39H34N6O6S2
40H37N5O7S2
39H36N5O6S2F
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
CH2CH2C6H5
p-F-C6H4
p-Cl-C6H4
p-I-C6H4
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
CH
N
N
N
98-100
110-112
oil
95-97
p-CH3-C6H4
p-OCH3-C6H4
p-NO2-C6H4
p-CN-C6H4
p-CH3CO-C6H4
p-F-C6H4CH2
p-F-C6H4CO
p-NO2-C6H4CH2
o-F-C6H4
70-72
85-87
77-79
124-127
82-84
103-105
124-126
145-147
133-135
84-86
C39H34N5O7S2F
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
39H36N6O8S2
38H34N5O6S2F
38H34N5O6S2Cl
39H37N5O6S2
39H37N5O7S2
38H34N5O6S2Cl
39H34N5O6S2F3
40H39N5O6S2
38H33N5O6S2Cl2
37H34N6O6S2
36H33N7O6S2
40H38N4O6S2
37H32N5O6S2F
38H35N5O6S2
38H36N6O6S2
o-Cl-C6H4
o-CH3-C6H4
o-OCH3-C6H4
m-Cl-C6H4
m-CF3-C6H4
o,m-2CH3-C6H3
m,p-2Cl-C6H3
2-pyridinyl
84-86
85-87
138-140
81-83
174-176
130-132
183-185
187-189
131-133
145-147
132-134
80-82
2-pyrimidinyl
CH2C6H5
p-F-C6H4
o-CH3-C6H4
p-NH2-C6H4
H
CH3
a
b
Yield of synthesized compounds after purification by column chromatography. All compounds were analyzed for C, H, N. Analytical
results were within 0.4% of theoretical values.
Ta ble 2. Activities of Synthesized Compounds 61-87 and
For the most potent compound in this series (deriva-
tive 63), placement of fluorine in the meta position
(compound 74) resulted in a 14-fold decrease in potency.
The same effect was not observed for derivatives 66 and
76, which possess methyl groups in the para and ortho
positions, respectively, and showed the same potency.
A potency increase was also observed with compounds
66 and 68, containing nitro and methyl groups in the
para position of the phenyl ring, which are 4- and 10-
fold more active than KN-62, respectively. Addition of
electron-withdrawing groups, such as nitrile (69) and
acetyl (70), and electron-donating groups, such as meth-
oxy (67) and amino (87), results in a decrease in potency
(from 2- to 3-fold). In derivative 67, when the methoxy
group was placed in the ortho position (compound 77),
a 2-fold increase in potency was observed.
These results support the suggestion that the phenyl-
piperazine moiety interacts with the active site of
the receptor and indicate that the compound/receptor
interaction can be improved by the presence of
adequate chemical groups in the para position of the
phenyl ring
For the compound 80, the dramatic reduction of
activity due to the insertion of a second methyl group
in the phenyl ring could be a result of the steric
bulkiness of the o,m-xylyl residue, which may prevent
interaction with the receptor.
KN-62 on the Calcium Influx in Human Monocytes
a
a
IC50 ( SE
IC50 ( SE
compd
(nM)
compd
(nM)
KN-62 (1)
51.1 ( 1.1
21.1 ( 0.8
600 ( 27
1.3 ( 0.1
105 ( 1.1
176 ( 3.8
13.5 ( 0.2
132 ( 4.3
5.8 ( 0.6
97.7 ( 2.2
70.8 ( 0.8
5.8 ( 0.5
380 ( 8.8
12.3 ( 0.3
74
75
76
77
78
79
80
81
82
83
84
85
86
87
18.5 ( 0.4
15.8 ( 0.9
15.1 ( 1.4
84.1 ( 5
61
62
63
64
65
66
67
68
69
70
71
72
73
14.1 ( 0.3
31.1 ( 1.2
1122 ( 25
33.9 ( 0.8
170 ( 3.9
79.8 ( 0.7
65.3 ( 0.8
6.0 ( 0.3
28.8 ( 0.3
101 ( 1.3
a
IC50 ) 50% inhibitory concentration, which represents the
mean from dose-response curves of at least three experiments.
All experiments were repeated three times.
increasing size of the halogen atom. Starting from
compound 64, placement of chloro in the meta (com-
pound 78) or ortho (compound 75) position led to a 6-fold
increase of potency. The same derivatives 75 and 78
were also 3-fold more potent than KN-62. In the same
compound 64, the introduction of a second chloro atom
in the meta position (compound 81) led to a 3-fold
increase of activity.

1322 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Baraldi et al.
F igu r e 3. Dose-dependent inhibition of ATP-dependent IL-
1â release by KN-62 and its derivatives. Cells were incubated
in serum-free RPMI, pretreated with LPS, and stimulated with
ATP as described in Materials and Methods. The P2X7
antagonists were added 5 min before ATP. Supernatants were
withdrawn 30 min after ATP addition and assayed for IL-1â
content. Experimental points are averages of quadruplicate
determinations.
F igu r e 2. Effect of KN-62 and its derivatives on ethidium
bromide uptake. P2X7-transduced HEK293 cells were incu-
bated in a fluorometer cuvette in a modified (no Ca2+, 500 mM
EGTA added) standard saline solution (see Materials and
Methods). The P2X7 antagonists were added 5 min before ATP.
Uptake inhibition is expressed as a percentage of maximal
ethidium bromide (EB) uptake rate (arbitrary fluorescence
units per min) measured in the absence of inhibitor. Experi-
mental points are averages of triplicate determinations.
was a small residual cytokine secretion even at concen-
trations exceeding 100 nM. Incubation of macrophages
with the KN-62 derivatives throughout the IL-1â release
experiments had no detrimental effects on cell viability,
as judged by the lack of morphological alterations
(shrinking or swelling), loss of adherence, or release of
cytosolic enzymes (not shown), thus indicating that
these compounds are good tools for the investigation of
inflammatory cell functions in vitro. None of the com-
pounds tested on IL-1â release had any effect on isolated
CaMII kinase activity up to 20 µM (not shown).
The introduction of a methylene spacer separating the
piperazine nitrogen and the p-fluorinephenyl ring fur-
nished a compound (71) that was 4-fold less active than
the parent compound 63. The same effect was observed
comparing the activities of 68 and 73, where the
insertion of a methylene unit decreases the potency by
2-fold.
From a comparison of the activities of compounds 63
and 65 with that of 76 and 86, respectively, a slight
change in potency was observed by removal of the
methyl on the nitrogen on the R-position of the tyrosine
moiety, and this confirms that the presence of this
substituent was not fundamental for antagonistic activ-
ity.
In conclusion, the structure-activity relationships
(SAR) showed that the basicity of the piperazine nitro-
gen linked to the phenyl-substituted ring was essential
for activity (compounds 63 and 71 vs 72). This is
confirmed by replacement of the piperazine moiety with
a piperidine (61 vs 84), which led to a compound with
reduced activity. The most potent P2X7 receptor an-
tagonist identified in this study (compound 63) con-
tained a p-fluorine substituent on the phenylpiperazine
moiety. Figure 2 shows the effect of three of the most
potent compounds (63, 71, and 85) on ethidium bromide
uptake, a reliable index of activation of the P2X7
receptor/pore.
We then tested the activity of the four most potent
KN-62 derivatives on ATP-stimulated secretion of IL-
1â from monocyte-derived human macrophages, a key
cell type in inflammation and native immunity. Figure
3 shows that the KN-62 IC50 for IL-1â was lower than
that for Ca influx (20 vs 51 nM). Two of the compounds
tested, 71 and 85, had an IC50 lower than KN-62 (3.0
and 2.5 nM, respectively).
Exp er im en ta l Section
Ch em ica l Ma ter ia ls a n d Meth od s. Gen er a l P r oced u r e.
All reactions were carried out under an inert atmosphere of
dry nitrogen unless otherwise described. Standard hypodermic
syringe (glass/metal Luer) techniques were applied for trans-
ferring dry solvents. Starting materials were purchased and
used without any purification. Reaction courses and product
mixtures were routinely monitored by TLC on silica gel
(precoated F254 Macherey-Nagel plates) and visualized with
aqueous KMnO4. Melting points (mp) were determined on a
Buchi-Tottoli apparatus and are uncorrected. Optical rotations
were measured on a Perkin-Elmer 241 polarimeter. Chemical
shifts (δ) are given in ppm upfield from tetramethylsilane. All
1
products reported showed H NMR spectra in agreement with
the assigned structures. 1H NMR was determined in CDCl3
solution with a Bruker AC200 spectrometer. Microanalytical
analyses were conducted by the Mycroanalytical Laboratory
of the Chemistry Department of the University of Ferrara.
Organic solutions were dried over anhydrous Na2SO4. Metha-
nol was distilled from magnesium turnings, dioxan was
distilled from calcium hydride, and dry DMF was distilled from
calcium chloride and stored over molecular sieves (3 Å). In
high-pressure hydrogenation experiments, a Parr shaker on
a high-pressure autoclave was used.
Gen er a l P r oced u r e A for th e Syn th esis of Com p ou n d s
9-34. To a solution of 7a or 7b (1 mmol) in dry DMF (5 mL)
cooled to 0 °C was added EDC (211 mg, 1.1 mmol, 1.1 equiv),
HOBt (1.1 mmol), and a suitable N-substituted piperazine (1.1
mmol).29 This mixture was stirred for 24 h and then concen-
Interestingly, 71 and 85 caused a complete inhibition
of IL-1â release, while with KN-62, 63, and 68, there

Analogues of KN-62
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1323
trated in vacuo. The residue was dissolved in EtOAc (10 mL)
and was washed with water (5 mL) and then with brine (5
mL). The organic layer was dried (Na2SO4) and concentrated
in vacuo. The residue purified by column chromatography
using EtOAc/petroleum ether (1:1, v/v) as eluent furnished the
derivatives 9-34.
1-[(S)-N-ter t-Bu tyloxycar bon yl-N-m eth yltyr osyl]-4-ben -
zylp ip er a zin e (9). Following general procedure A, this prod-
uct was obtained as a white solid (yield 98%): mp 104-106
°C, [R] ) -63.7, c ) 0.91% in CHCl3. 1H NMR (CDCl3) δ: 1.36
(s, 9H), 2.28 (m, 4H), 2.79 (s, 3H), 2.83 (s, 2H), 2.89 (dd, J )
16.7 and 7.3 Hz, 2H), 3.49 (m, 4H), 5.21 (t, J ) 7.3 Hz, 1H),
5.85 (s, 1H), 6.72 (d, J ) 8.3 Hz, 2H), 7.05 (d, J ) 8.3 Hz, 2H),
7.20 (m, 5H).
1-[(S)-N-ter t-Bu t yloxyca r b on yl-N-m e t h ylt yr osyl]-4-
p h en eth ylp ip er a zin e (10). Following general procedure A,
this product was obtained as a white solid (yield 46%): mp
147-150 °C; [R] ) -46.8, c ) 1.5% in CH2Cl2. 1H NMR (CDCl3)
δ: 1.37 (s, 9H), 2.38 (m, 4H), 2.58 (m, 2H), 2.75 (m, 2H), 2.81
(s, 3H), 2.89 (dd, J ) 14.1 and 8.3 Hz, 2H), 3.49 (m, 4H), 5.21
(t, J ) 8.3 Hz, 1H), 5.49 (s, 1H), 6.70 (d, J ) 8.3 Hz, 2H), 7.00
(d, J ) 8.3 Hz, 2H), 7.23 (m, 5H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
a cetylp h en yl)p ip er a zin e (18). Following general procedure
A, this product was obtained as a yellow solid (yield 67%): mp
123-125 °C; [R] ) -36.2, c ) 0.5% in CHCl3. 1H NMR (CDCl3)
δ: 1.34 (s, 9H), 2.53 (s, 3H), 2.84 (s, 3H), 2.93 (dd, J ) 14.8
and 7.3 Hz, 2H), 3.42 (m, 4H), 3.54 (m, 4H), 5.26 (t, J ) 7.3
Hz, 1H), 5.91 (s, 1H), 6.77 (dd, J ) 8.0 and 7.8 Hz, 4H), 7.10
(d, J ) 8.0 Hz, 2H), 7.87 (d, J ) 8.0 Hz, 2H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
flu or oben zyl)p ip er a zin e (19). Following general procedure
A, this product was obtained as a brown oil (yield 78%): [R] )
1
-62.2, c ) 0.49% in CHCl3. H NMR (CDCl3) δ: 1.37 (s, 9H),
2.37 (m, 4H), 2.84 (s, 5H), 2.94 (m, 1H), 3.11 (m, 1H), 3.53 (m,
4H), 4.57 (m, 1H), 5.16 (t, J ) 7.3 Hz, 1H), 6.78 (d, J ) 8.1
Hz, 2H), 7.00 (dd, J ) 8.2 and 8.1 Hz, 4H), 7.21 (d, J ) 8.2
Hz, 2H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
flu or oben zoyl)p ip er a zin e (20). Following general procedure
A, this product was obtained as a white oil (yield 97%): [R] )
1
-10.6, c ) 1.18% in CH2Cl2. H NMR (CDCl3) δ: 1.38 (s, 9H),
2.85 (s, 3H), 3.00 (m, 2H), 3.50 (m, 8H), 4.57 (m, 1H), 5.16 (m,
1H), 6.73 (d, J ) 8.3 Hz, 2H), 7.08 (m, 4H), 7.38 (d, J ) 8.2
Hz, 2H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
flu or op h en yl)p ip er a zin e (11). Following general procedure
A, this product was obtained as a white solid (yield 94%): mp
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
n itr oben zyl)p ip er a zin e (21). Following general procedure
A, this product was obtained as a brown oil (yield 78%): [R] )
-30, c ) 0.46% in CHCl3. 1H NMR (CDCl3) δ: 1.35 (s, 9H),
2.35 (m, 4H), 2.82 (s, 2H), 2.89 (m, 2H), 2.96 (s, 3H), 3.51 (m,
4H), 4.88 (m, 1H), 5.19 (m, 1H), 6.75 (d, J ) 8.5 Hz, 2H), 7.05
(d, J ) 8.5 Hz, 2H), 7.46 (d, J ) 8.3 Hz, 2H), 8.14 (d, J ) 8.3
Hz, 2H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(1-
flu or op h en yl)p ip er a zin e (22). Following general procedure
A, this product was obtained as a white solid (yield 78%): mp
73-75 °C; [R] ) -77, c ) 0.76% in CHCl3. 1H NMR (CDCl3) δ:
1.38 (s, 9H), 2.84 (s, 3H), 2.94 (m, 4H), 3.11 (dd, J ) 13.8 and
7.6 Hz, 2H), 3.58 (m, 4H), 5.26 (t, J ) 7.2 Hz, 1H), 6.76 (m,
3H), 7.01 (m, 5H), 9.46 (s, 1H).
1
67-69 °C; [R] ) -85.4, c ) 1.29% in CHCl3. H NMR (CDCl3)
δ: 1.34 (s, 9H), 2.78 (s, 3H), 2.89 (dd, J ) 16.7 and 7.3 Hz,
2H), 3.07 (m, 4H), 3.50 (m, 4H), 5.21 (t, J ) 7.3 Hz, 1H), 5.85
(s, 1H), 6.86 (m, 8H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
ch lor op h en yl)p ip er a zin e (12). Following general procedure
A, this product was obtained as a white solid (yield 78%): mp
73-75 °C; [R] ) -77, c ) 0.76% in CHCl3. 1H NMR (CDCl3) δ:
1.39 (s, 9H), 1.56 (m, 2H), 2.89 (s, 3H), 2.95 (m, 4H), 3.54 (m,
4H), 5.25 (t, J ) 7.2 Hz, 1H), 6.78 (m, 3H), 7.02 (d, J ) 8.4
Hz, 1H), 7.09 (d, J ) 8.2 Hz, 2H), 7.22 (m, 2H), 9.46 (s, 1H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
iod op h en yl)p ip er a zin e (13). Following general procedure
A, this product was obtained as a yellow solid (yield 92%): mp
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(2-
ch lor op h en yl)p ip er a zin e (23). Following general procedure
A, this product was obtained as a white solid (yield 98%): mp
1
73-75 °C; [R] ) -65.4, c ) 1.02% in CHCl3. H NMR (CDCl3)
δ: 1.38 (s, 9H), 2.82 (s, 3H), 2.93 (dd, J ) 14.6 and 7.2 Hz,
2H), 3.12 (m, 4H), 3.51 (m, 4H), 5.24 (t, J ) 7.2 Hz, 1H), 5.82
(s, 1H), 6.61 (d, J ) 8.8 Hz, 2H), 6.72 (d, J ) 8.4 Hz, 2H), 7.10
(d, J ) 8.4 Hz, 2H), 7.52 (d, J ) 8.8 Hz, 2H).
1
70-72 °C; [R] ) -70.3, c ) 1.15% in CHCl3. H NMR (CDCl3)
δ: 1.38 (s, 9H), 2.84 (s, 3H), 2.91 (m, 6H), 3.71 (m, 4H), 5.25
(t, J ) 7.2 Hz, 1H), 5.82 (s, 1H), 6.74 (m, 2H), 7.04 (m, 5H),
7.34 (d, J ) 9.2 Hz, 1H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(p-
tolyl)p ip er a zin e (14). Following general procedure A, this
product was obtained as an oil (yield 93%): [R] ) -83.3, c )
1.34% in CHCl3. 1H NMR (CDCl3) δ: 1.38 (s, 9H), 2.27 (s, 3H),
2.81 (s, 3H), 2.86 (dd, J ) 14 and 7.6 Hz, 2H), 3.42 (m, 4H),
3.54 (m, 4H), 5.26 (t, J ) 7 Hz, 1H), 6.80 (m, 4H), 7.06 (m,
4H), 9.5 (s, 1H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
m eth oxyp h en yl)p ip er a zin e (15). Following general proce-
dure A, this product was obtained as a foamy yellow solid (yield
89%): [R] ) -63.3, c ) 1.1% in CHCl3. 1H NMR (CDCl3) δ:
1.38 (s, 9H), 2.82 (s, 3H), 2.87 (m, 6H), 3.52 (m, 4H), 3.77 (s,
3H), 5.26 (t, J ) 7.2 Hz, 1H), 6.71 (d, J ) 8.4 Hz, 2H), 6.84
(m, 2H), 7.02 (d, J ) 8.6 Hz, 2H), 7.10 (d, J ) 8.6 Hz, 2H),
9.46 (s, 1H).
1-[(S)-N-ter t-Bu t yloxyca r bon yl-N-m et h ylt yr osyl]-4-o-
tolylp ip er a zin e (24). Following general procedure A, this
product was obtained as a white solid (yield 95%): mp 80-82
1
°C; [R] ) -66.7, c ) 1.9% in CHCl3. H NMR (CDCl3) δ: 1.38
(s, 9H), 2.28 (s, 3H), 2.74 (m, 6H), 2.86 (s, 3H), 3.65 (m, 4H),
5.26 (t, J ) 7.2 Hz, 1H), 6.01 (s, 1H), 6.74 (m, 2H), 7.10 (m,
6H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(2-
m eth oxyp h en yl)p ip er a zin e (25). Following general proce-
dure A, this product was obtained as a white solid (yield
1
97%): mp 153-155 °C; [R] ) -126.9, c ) 0.93% in CHCl3. H
NMR (CDCl3) δ: 1.38 (s, 9H), 2.83 (s, 3H), 2.93 (m, 6H), 3.64
(m, 4H), 3.86 (s, 3H), 5.27 (t, J ) 7.2 Hz, 1H), 5.82 (s, 1H),
6.72 (m, 3H), 6.89 (m, 2H), 7.06 (m, 3H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(3-
ch lor op h en yl)p ip er a zin e (26). Following general procedure
A, this product was obtained as a yellow solid (yield 93%): mp
60-62 °C; [R] ) -72, c ) 0.88% in CHCl3. 1H NMR (CDCl3) δ:
1.36 (s, 9H), 2.83 (s, 3H), 2.99 (m, 6H), 3.52 (m, 4H), 5.26 (t, J
) 7.2 Hz, 1H), 6.76 (m, 4H), 7.12 (m, 4H), 9.46 (s, 1H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(3-
tr iflu or om eth ylp h en yl)p ip er a zin e (27). Following general
procedure A, this product was obtained as a yellow solid (yield
78%): mp 102-104 °C; [R] ) -63.4, c ) 0.8% in CHCl3. 1H
NMR (CDCl3) δ: 1.39 (s, 9H), 2.83 (s, 3H), 3.04 (m, 6H), 3.56
(m, 4H), 5.25 (t, J ) 7.2 Hz, 1H), 5.82 (s, 1H), 6.73 (d, J ) 8.3
Hz, 2H), 7.02 (m, 3H), 7.10 (d, J ) 8.3 Hz, 2H), 7.35 (m, 1H).
1-[(S)-N-ter t-Bu tyloxycar bon yl-N-m eth yltyr osyl]-4-(2,3-
d im eth ylp h en yl)p ip er a zin e (28). Following general proce-
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
n itr op h en yl)p ip er a zin e (16). Following general procedure
A, this product was obtained as a yellow solid (yield 88%): mp
211-213 °C; [R] ) -66.7, c ) 1.02% in CHCl3. 1H NMR (CDCl3)
δ: 1.39 (s, 9H), 2.74 (dd, J ) 14 and 7.6 Hz, 2H), 2.84 (s, 3H),
3.34 (m, 4H), 3.56 (m, 4H), 5.25 (t, J ) 7.2 Hz, 1H), 6.71 (d, J
) 3.4 Hz, 2H), 6.76 (d, J ) 3.4 Hz, 2H), 7.12 (d, J ) 8.4 Hz,
2H), 8.12 (d, J ) 9.2 Hz, 2H), 9.5 (s, 1H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-(4-
cya n op h en yl)p ip er a zin e (17). Following general procedure
A, this product was obtained as an oil (yield 76%): [R] ) -49.7,
c ) 1.47% in CHCl3. 1H NMR (CDCl3) δ: 1.39 (s, 9H), 1.56 (m,
2H), 2.84 (s, 3H), 3.10 (m, 4H), 3.52 (m, 4H), 5.31 (t, J ) 7.2
Hz, 1H), 6.73 (d, J ) 8.6 Hz, 2H), 6.79 (d, J ) 9.2 Hz, 2H),
7.11 (d, J ) 8.2 Hz, 2H), 7.48 (d, J ) 8.8 Hz, 2H), 9.46 (s, 1H).

1324 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
dure A, this product was obtained as an oil (yield 91%): [R] )
Baraldi et al.
general procedure B, this product was obtained as a white solid
(yield 82%): mp 58-60 °C; [R] ) -33.8, c ) 0.73% in CH2Cl2.
1H NMR (CDCl3) δ: 1.39 (s, 9H), 2.05 (s, 1H), 2.17 (s, 1H),
2.39 (m, 3H), 2.57 (m, 3H), 2.72 (m, 4H), 2.87 (m, 2H), 3.36
(m, 2H), 3.51 (m, 1H), 5.15 (t, J ) 7 Hz, 1H), 6.77 (m, 2H),
7.03 (d, J ) 8.3 Hz, 2H), 7.18 (m, 3H), 7.25 (m, 2H), 7.59 (t, J
) 8.0 Hz, 1H), 8.25 (t, J ) 5.8 Hz, 2H), 8.53 (d, J ) 6.2 Hz,
1H), 8.79 (d, J ) 6.2 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-flu or op h en yl)p ip er a zin e (37). Fol-
lowing general procedure B, this product was obtained as a
oil (yield 85%): [R] ) -54.1, c ) 1.31% in CHCl3. 1H NMR
(CDCl3) δ: 1.33 (s, 9H), 2.73 (s, 3H), 2.91 (m, 6H), 3.51 (m,
4H), 5.17 (t, J ) 7.2 Hz, 1H), 6.77 (t, J ) 8.6 Hz, 4H), 7.01 (t,
J ) 8.6 Hz, 4H), 7.62 (t, J ) 7.6 Hz, 1H), 8.26 (t, J ) 7.6, 2H),
8.52 (d, J ) 6.1 Hz, 1H), 8.79 (d, J ) 6.1 Hz, 1H), 9.40 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-ch lor op h en yl)p ip er a zin e (38). Fol-
lowing general procedure B, this product was obtained as a
white liquid (yield 86%): [R] ) -68.4, c ) 1% in CHCl3. 1H
NMR (CDCl3) δ: 1.33 (s, 9H), 1.57 (m, 2H), 2.74 (s, 3H), 3.09
(m, 4H), 3.48 (m, 4H), 5.22 (t, J ) 7 Hz, 1H), 6.77 (m, 4H),
7.10 (t, J ) 8.6 Hz, 2H), 7.23 (m, 2H), 7.61 (t, J ) 8.6 Hz, 1H),
8.25 (dd, J ) 6.6 and 6.2 Hz, 2H), 8.54 (d, J ) 6.2 Hz, 1H),
8.80 (d, J ) 6.2 Hz, 1H), 9.41 (s, 1H).
1
-20.3, c ) 0.7% in CH2Cl2. H NMR (CDCl3) δ: 1.44 (s, 9H),
2.18 (s, 3H), 2.26 (s, 3H), 2.85 (s, 3H), 2.93 (m, 6H), 3.65 (m,
4H), 5.25 (t, J ) 7.2 Hz, 1H), 5.82 (s, 1H), 6.76 (m, 3H), 6.94
(m, 1H), 7.06 (m, 3H).
1-[(S)-N-ter t-Bu tyloxycar bon yl-N-m eth yltyr osyl]-4-(3,4-
d ich lor op h en yl)p ip er a zin e (29). Following general proce-
dure A, this product was obtained as a white solid (yield
85%): mp 77-78 °C; [R] ) -71.4, c ) 0.42% in CHCl3. 1H NMR
(CDCl3) δ: 1.39 (s, 9H), 2.84 (s, 3H), 2.99 (m, 6H), 3.56 (m,
4H), 5.25 (t, J ) 7.2 Hz, 1H), 6.75 (m, 2H), 6.88 (m, 1H), 7.08
(m, 2H), 7.26 (m, 2H), 9.46 (s, 1H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-p y-
r id in -2-ylp ip er a zin e (30). Following general procedure A,
this product was obtained as a white oil (yield 89%): [R] )
1
-57.1, c ) 0.7% in CH2Cl2. H NMR (CDCl3) δ: 1.39 (s, 9H),
2.83 (s, 3H), 2.97 (d, J ) 7.6 Hz, 1H), 3.50 (m, 9H), 4.95 (m,
1H), 5.26 (t, J ) 7.5 Hz, 1H), 6.66 (m, 4H), 7.01 (d, J ) 8.2
Hz, 1H), 7.08 (d, J ) 8.4 Hz, 1H), 7.49 (m, 1H), 8.18 (m, 1H).
1-[(S)-N-ter t-Bu tyloxyca r bon yl-N-m eth yltyr osyl]-4-p y-
r im id in -2-ylp ip er a zin e (31). Following general procedure A,
this product was obtained as a white solid (yield 91%): mp
109-111 °C; [R] ) -54.5, c ) 0.27% in CH2Cl2. 1H NMR
(CDCl3) δ: 1.38 (s, 9H), 2.83 (s, 3H), 3.02 (m, 2H), 3.53 (m,
5H), 3.88 (m, 3H), 4.92 (m, 1H), 5.26 (t, J ) 7.5 Hz, 1H), 6.53
(m, 1H), 6.72 (m, 2H), 7.04 (d, J ) 7.8 Hz, 1H), 7.11 (d, J )
7.8 Hz, 1H), 8.32 (m, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-iod op h en yl)p ip er a zin e (39). Fol-
lowing general procedure B, this product was obtained as a
yellow oil (yield 98%): [R] ) -48.8, c ) 1.22% in CHCl3. 1H
NMR (CDCl3) δ: 1.34 (s, 9H), 2.73 (s, 3H), 2.98 (m, 6H), 3.51
(m, 4H), 5.21 (t, J ) 7 Hz, 1H), 6.64 (d, J ) 8.8, 2H), 6.76 (t,
J ) 8.6, 2H), 7.09 (t, J ) 8.6, 2H), 7.53 (d, J ) 8.8, 2H), 7.61
(t, J ) 7.2 Hz, 1H), 8.25 (t, J ) 7.2 Hz, 2H), 8.53 (d, J ) 6.1
Hz, 1H), 8.80 (d, J ) 6.1 Hz, 1H), 9.41 (s, 1H).
1-[(S)-N-ter t-Bu tyloxycar bon yl-N-m eth yltyr osyl]-4-ben -
zylp ip er id in e (32). Following general procedure A, this
product was obtained as a white solid (yield 97%): mp 70-72
°C; [R] ) -61.7, c ) 0.46% in CH2Cl2. 1H NMR (CDCl3) δ: 1.39
(s, 9H), 1.62 (m, 5H), 2.50 (m, 3H), 2.86 (dd, J ) 12.6 and 6.8
Hz, 6H), 3.95 (m, 1H), 4.58 (m, 1H), 5.21 (t, J ) 6.8 Hz, 1H),
5.85 (s, 1H), 6.72 (d, J ) 7.5 Hz, 2H), 7.10 (m, 4H), 7.25 (m,
3H).
1-[(S )-N -t er t -Bu t yloxyca r b on ylt yr osyl]-4-(4-flu or o-
p h en yl)p ip er a zin e (33). Following general procedure A, this
product was obtained as a yellow oil (yield 87%): [R] ) +7.0,
c ) 0.43% in CH2Cl2. 1H NMR (CDCl3) δ: 1.43 (s, 9H), 2.93
(m, 6H), 3.57 (m, 4H), 4.82 (t, J ) 8.6 Hz, 1H), 5.45(s, 1H),
6.75 (m, 4H), 6.93 (d, J ) 8.4 Hz, 2H), 7.04 (d, J ) 8.4 Hz,
2H).
1-[(S)-N-ter t-Bu tyloxyca r bon yltyr osyl]-4-o-tolylp ip er a -
zin e (34). Following general procedure A, this product was
obtained as a white solid (yield 94%): mp 80-82 °C; [R])+14.9,
c ) 1.03% in CH2Cl2. 1H NMR (CDCl3) δ: 1.43 (s, 9H), 2.26 (s,
3H), 2.31 (m, 1H), 2.69 (m, 3H), 2.92 (d, J ) 6.9 Hz, 2H), 3.29
(m, 1H), 3.46 (m, 1H), 3.69 (m, 2H), 4.85 (dd, J ) 15.7 and 7.3
Hz, 1H), 5.47 (d, J ) 8.6 Hz, 1H), 6.20 (s, 1H), 6.74 (d, J ) 8.4
Hz, 2H), 6.86 (d, J ) 7.7 Hz, 1H), 7.03 (t, J ) 8.4 Hz, 3H),
7.15 (t, J ) 7.4 Hz, 2H).
Gen er a l P r oced u r e B for th e Syn th esis of Com p ou n d s
35-60. To a suspension of NaH (24 mg of 55-65% oil
suspension, 0.6 mmol, 1.2 equiv) in dry THF (5 mL) was added
9-34 (0.5 mmol, 1 equiv). After 10 min, the isoquinolinesulfo-
nyl chloride (1 mmol, 2 equiv) dissolved in dry DCM (2 mL)
was added. This mixture was stirred for 18 h at room
temperature and then concentrated in vacuo. The residue was
dissolved in a mixture of EtOAc (10 mL) and saturated
aqueous NaHCO3 (5 mL). After the layers were separated, the
organic layer was dried (Na2SO4) and concentrated in vacuo,
and the residue purified by column chromatography using
EtOAc/MeOH (9.8:0.2, v/v) as eluent yielded the derivatives
35-60.
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-tolylp ip er a zin e (40). Following general
procedure B, this product was obtained as a yellow oil (yield
1
90%): [R] ) -10.9, c ) 1.34% in CHCl3. H NMR (CDCl3) δ:
1.33 (s, 9H), 2.05 (s, 3H), 2.74 (s, 3H), 2.82 (m, 2H), 3.04 (m,
4H), 3.51 (m, 4H), 5.22 (t, J ) 7.2 Hz, 1H), 6.79 (t, J ) 8.4 Hz,
4H), 7.09 (d, J ) 8.4 Hz, 4H), 7.61 (t, J ) 7.6 Hz, 1H), 8.26
(m, 2H), 8.53 (d, J ) 6 Hz, 1H), 8.80 (d, J ) 6.2 Hz, 1H), 9.41
(s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m et h ylt yr osyl]-4-(4-m et h oxyp h en yl)p ip er a zin e (41).
Following general procedure B, this product was obtained as
1
a yellow oil (yield 82%): [R] ) -63.3, c ) 1.1% in CHCl3. H
NMR (CDCl3) δ: 1.33 (s, 9H), 1.57 (m, 2H), 2.73 (s, 3H), 2.96
(m, 4H), 3.51 (m, 4H), 3.76 (s, 3H), 5.21 (t, J ) 7 Hz, 1H), 6.82
(m, 4H), 7.10 (t, J ) 8.6 Hz, 2H), 7.23 (m, 2H), 7.61 (t, J ) 8.6
Hz, 1H), 8.25 (dd, J ) 6.6 and 6.2 Hz, 2H), 8.54 (d, J ) 6.2
Hz, 1H), 8.78 (d, J ) 6.2 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-n itr op h en yl)p ip er a zin e (42). Fol-
lowing general procedure B, this product was obtained as a
yellow solid (yield 79%): mp 46-47 °C; [R] ) -58.71, c ) 1.01%
1
in CHCl3. H NMR (CDCl3) δ: 1.34 (s, 9H), 2.83 (dd, J ) 14
and 6.4 Hz, 2H), 2.95 (s, 3H), 3.44 (m, 4H), 3.56 (m, 4H), 5.19
(t, J ) 7.2 Hz, 1H), 6.79 (dd, J ) 7.2 and 3.6 Hz, 4H), 7.10 (d,
J ) 8.6 Hz, 2H), 7.61 (t, J ) 7.8 Hz, 1H), 8.11 (d, J ) 7.4 Hz,
2H), 8.25 (t, J ) 8.8 Hz, 2H), 8.51 (d, J ) 6.2 Hz, 1H), 8.80 (d,
J ) 6.2 Hz, 1H), 9.51 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-cya n op h en yl)p ip er a zin e (43). Fol-
lowing general procedure B, this product was obtained as a
yellow oil (yield 97%): [R] ) +4.21, c ) 1.4% in CHCl3. 1H
NMR (CDCl3) δ: 1.34 (s, 9H), 2.74 (s, 3H), 3.09 (m, 6H), 3.49
(m, 4H), 5.22 (t, J ) 7 Hz, 1H), 6.79 (t, J ) 8.6 Hz, 4H), 7.11
(t, J ) 8.4 Hz, 3H), 7.51 (d, J ) 8.6 Hz, 1H), 7.63 (t, J ) 7.6
Hz, 1H), 8.26 (dd, J ) 6.8 and 4.4 Hz, 2H), 8.51 (d, J ) 6 Hz,
1H), 8.80 (d, J ) 6 Hz, 1H), 9.41 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-ben zylp ip er a zin e (35). Following gen-
eral procedure B, this product was obtained as a yellow oil
(yield 90%): [R] ) -64.4, c ) 1.15% in CHCl3. 1H NMR (CDCl3)
δ: 1.31 (s, 9H), 2.72 (m, 4H), 2.90 (m, 2H), 3.48 (m, 7H), 3.70
(m, 2H), 5.15 (t, J ) 7 Hz, 1H), 6.76 (t, J ) 8.6 Hz, 3H), 7.05
(t, J ) 8.6 Hz, 3H), 7.28 (m, 3H), 7.63 (m, 1H), 8.27 (m, 2H),
8.54 (d, J ) 6.1 Hz, 1H), 8.79 (d, J ) 6.1 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-p h en eth ylp ip er a zin e (36). Following
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-a cetylp h en yl)p ip er a zin e (44). Fol-
lowing general procedure B, this product was obtained as an

Analogues of KN-62
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1325
1
oil (yield 98%): [R] ) -51.82, c ) 1.2% in CHCl3. 1H NMR
(CDCl3) δ: 1.35 (s, 9H), 2.53 (s, 3H), 2.74 (s, 3H), 3.49 (m, 10H),
5.19 (t, J ) 7.2 Hz, 1H), 6.77 (t, J ) 8.6, 2H), 6.84 (t, J ) 8.9
Hz, 2H), 7.10 (t, J ) 8.6 Hz, 2H), 7.62 (t, 1H), 7.89 (d, J ) 8.9
Hz, 2H), 8.28 (d, J ) 6.2 Hz, 2H), 8.51 (d, J ) 6.2 Hz, 1H),
8.80 (d, J ) 6.2 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-flu or oben zyl)p ip er a zin e (45). Fol-
lowing general procedure B, this product was obtained as a
brown oil (yield 77%): [R] ) -38.9, c ) 0.66% in CHCl3. 1H
NMR (CDCl3) δ: 1.32 (s, 9H), 2.72 (s, 3H), 2.90 (m, 3H), 3.50
(m, 7H), 3.73 (m, 2H), 5.15 (t, J ) 7 Hz, 1H), 6.78 (t, J ) 8.6
Hz, 2H), 7.03 (t, J ) 8.6 Hz, 4H), 7.25 (t, J ) 6.6 Hz, 2H), 7.64
(m, 1H), 8.28 (d, J ) 7.8 Hz, 2H), 8.54 (d, J ) 6.1 Hz, 2H),
8.81 (d, J ) 6.2 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-flu or oben zoyl)p ip er a zin e (46). Fol-
lowing general procedure B, this product was obtained as a
white oil (yield 97%): [R] ) -27.4, c ) 0.74% in CH2Cl2. 1H
NMR (CDCl3) δ: 1.35 (s, 9H), 1.62 (m, 5H), 2.50 (m, 3H), 2.72
(s, 3H), 2.90 (m, 3H), 3.90 (m, 1H), 4.50 (m, 1H), 5.12 (t, J )
6.2 Hz, 1H), 6.76 (d, J ) 8.4 Hz, 2H), 7.08 (d, J ) 8.6 Hz, 4H),
7.23 (m, 3H), 7.67 (m, 1H), 8.27 (d, J ) 6.0 Hz, 2H), 8.54 (d, J
) 6.1 Hz, 1H), 8.80 (d, J ) 6.0 Hz, 1H), 9.42 (s, 1H).
in CHCl3. H NMR (CDCl3) δ: 1.35 (s, 9H), 2.73 (s, 3H), 2.92
(m, 6H), 3.51 (m, 4H), 5.21 (t, J ) 7 Hz, 1H), 6.79 (m, 5H),
7.14 (m, 4H), 7.61 (t, J ) 7.2 Hz, 1H), 8.26 (t, J ) 7.2 Hz, 1H),
8.53 (d, J ) 6.2 Hz, 1H), 8.80 (d, J ) 4.2 Hz, 1H), 9.41 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N -m e t h ylt yr osyl]-4-(3-t r iflu or om e t h ylp h e n yl)p ip e r a -
zin e (53). Following general procedure B, this product was
obtained as a white solid (yield 75%): mp 74-76 °C; [R] )
1
-48.9, c ) 0.76% in CHCl3. H NMR (CDCl3) δ: 1.35 (s, 9H),
2.74 (s, 3H), 3.01 (m, 6H), 3.65 (m, 4H), 5.20 (t, J ) 7 Hz, 1H),
6.79 (t, J ) 8.6 Hz, 2H), 7.08 (t, J ) 8.6 Hz, 5H), 7.37 (t, J )
7.8 Hz, 1H), 7.66 (t, J ) 7.8 Hz, 1H), 8.29 (dd, J ) 8.1 and 4.6
Hz, 2H), 8.58 (d, J ) 6.1 Hz, 1H), 8.81 (d, J ) 6.1 Hz, 1H),
9.44 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(2,3-d im eth ylp h en yl)p ip er a zin e (54).
Following general procedure B, this product was obtained as
a yellow oil (yield 44%): [R] ) -70, c ) 0.2% in CH2Cl2. 1H
NMR (CDCl3) δ: 1.39 (s, 9H), 2.21 (s, 3H), 2.27 (s, 3H), 2.76
(s, 3H), 2.82 (m, 6H), 3.58 (m, 4H), 5.20 (t, J ) 7 Hz, 1H), 6.80
(t, J ) 8.3 Hz, 3H), 6.95 (m, 1H), 7.08 (t, J ) 8.4 Hz, 3H), 7.66
(m, 1H), 8.26 (t, J ) 8.4, 2H), 8.55 (d, J ) 6.1 Hz, 1H), 8.81 (d,
J ) 6.1 Hz, 1H), 9.41 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl-4-(3,4-d ich lor op h en yl)p ip er a zin e (55).
Following general procedure B, this product was obtained as
a yellow solid (yield 52%): mp 85-87 °C; [R] ) +12, c ) 0.5%
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(4-n itr oben zyl)p ip er a zin e (47). Fol-
lowing general procedure B, this product was obtained as a
1
1
yellow oil (yield 54%): [R] ) -44.5, c ) 0.44% in CH3OH. H
in CHCl3. H NMR (CDCl3) δ: 1.34 (s, 9H), 2.73 (s, 3H), 2.92
NMR (CDCl3) δ: 1.30 (s, 9H), 2.73 (m, 4H), 2.90 (m, 2H), 3.50
(m, 7H), 3.70 (m, 2H), 5.15 (t, J ) 7 Hz, 1H), 6.79 (t, J ) 8.3
Hz, 2H), 7.06 (t, J ) 8.5 Hz, 2H), 7.49 (d, J ) 8.3 Hz, 2H),
7.65 (t, J ) 7.7 Hz, 1H), 8.18 (d, J ) 8.3 Hz, 2H), 8.29 (d, J )
7.7 Hz, 2H), 8.54 (d, J ) 6.0 Hz, 1H), 8.80 (d, J ) 6.1 Hz, 1H),
9.42 (s, 1H).
(m, 6H), 3.51 (m, 4H), 5.19 (t, J ) 7.0 Hz, 1H), 6.74 (m, 2H),
6.90 (s, 1H), 7.10 (d, J ) 7.4 Hz, 1H), 7.32 (d, J ) 7.4 Hz, 1H),
7.63 (t, J ) 7.6 Hz, 1H), 8.26 (dd, J ) 7 and 3.6 Hz, 1H), 8.54
(t, J ) 6 Hz, 1H), 8.81 (d, J ) 6 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-p yr id in -2-ylp ip er a zin e (56). Following
general procedure B, this product was obtained as a white solid
(yield 90%): mp 56-58 °C; [R] ) -59.3, c ) 0.43% in CH2Cl2.
1H NMR (CDCl3) δ: 1.35 (s, 9H), 2.75 (s, 3H), 3.00 (m, 1H),
3.45 (m, 8H), 3.85 (m, 1H), 5.19 (t, J ) 7 Hz, 1H), 6.69 (t, J )
8.4 Hz, 4H), 7.07 (t, J ) 8.4 Hz, 2H), 7.51 (t, J ) 6.6 Hz, 1H),
7.62 (m, 1H), 8.23 (t, J ) 7.1 Hz, 3H), 8.53 (d, J ) 6.1 Hz,
1H), 8.89 (d, J ) 6.2 Hz, 1H), 9.41 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-p yr im id in -2-ylp ip er a zin e (57). Fol-
lowing general procedure B, this product was obtained as a
white solid (yield 80%): mp 104-106 °C; [R] ) -48.6, c )
0.37% in CH2Cl2. 1H NMR (CDCl3) δ: 1.35 (s, 9H), 2.76 (s,
3H), 3.00 (m, 2H), 3.53 (m, 5H), 3.85 (m, 3H), 5.19 (t, J ) 7
Hz, 1H), 6.54 (m, 1H), 6.78 (t, J ) 8.4 Hz, 2H), 7.08 (t, J ) 8.4
Hz, 2H), 7.63 (m, 1H), 8.29 (m, 4H), 8.53 (d, J ) 6.1 Hz, 1H),
8.81 (d, J ) 6.2 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-ben zylp ip er id in e (58). Following gen-
eral procedure B, this product was obtained as a white solid
(yield 90%): mp 100-102 °C; [R] ) -39.3, c ) 0.4% in CH2-
Cl2. 1H NMR (CDCl3) δ: 1.31 (s, 9H), 2.72 (m, 4H), 2.90 (m,
2H), 3.48 (m, 7H), 3.70 (m, 2H), 5.15 (t, J ) 7 Hz, 1H), 6.76 (t,
J ) 8.6 Hz, 3H), 7.05 (d, J ) 8.6 Hz, 3H), 7.28 (m, 3H), 7.63
(m, 1H), 8.27 (d, J ) 7.8 Hz, 2H), 8.54 (d, J ) 6.1 Hz, 1H),
8.79 (d, J ) 6.1 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxyca r bon -
yltyr osyl]-4-(4-flu or op h en yl)p ip er a zin e (59). Following
general procedure B, this product was obtained as a white solid
(yield 79%): mp 87-89 °C; [R] ) -1.6, c ) 0.63% in CH2Cl2.
1H NMR (CDCl3) δ: 1.39 (s, 9H), 2.65 (m, 1H), 2.92 (m, 6H),
3.43 (m, 2H), 3.78 (m, 1H), 4.76 (m, 1H), 5.33 (d, 1H), 6.82 (m,
4H), 7.01 (m, 4H), 7.54 (t, J ) 7.6 Hz, 1H), 8.22 (dd, J ) 7.5
and 8.2 Hz, 2H), 8.53 (d, J ) 6.1 Hz, 1H), 8.80 (d, J ) 6.1 Hz,
1H), 9.41 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(1-flu or op h en yl)p ip er a zin e (48). Fol-
lowing general procedure B, this product was obtained as a
1
yellow oil (yield 98%): [R] ) -44.23, c ) 1.04% in CHCl3. H
NMR (CDCl3) δ: 1.34 (s, 9H), 2.75 (s, 3H), 2.92 (m, 6H), 3.52
(m, 4H), 5.22 (t, J ) 7 Hz, 1H), 6.79 (m, 5H), 7.14 (m, 4H),
7.62 (t, J ) 7.6 Hz, 1H), 8.26 (t, J ) 7.2 Hz, 1H), 8.53 (t, J )
6 Hz, 1H), 8.82 (d, J ) 6 Hz, 1H), 9.41 (s, 1H).
[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxyca r bon yl-
N-m eth yltyr osyl-4-(2-ch lor op h en yl)p ip er a zin e (49). Fol-
lowing general procedure B, this product was obtained as an
oil (yield 98%): [R] ) -40.6, c ) 1.23% in CHCl3. 1H NMR
(CDCl3) δ: 1.34 (s, 9H), 2.76 (s, 3H), 2.88 (m, 6H), 3.62 (m,
4H), 5.19 (t, J ) 7 Hz, 1H), 6.79 (t, J ) 8.6, 2H), 7.05 (dd, J )
6.2 and 8.6, 4H), 7.22 (d, J ) 6.2 Hz, 1H), 7.37 (d, J ) 7.9 Hz,
1H), 7.64 (t, 1H), 8.28 (t, J ) 7, 2H), 8.57 (d, J ) 6.1 Hz, 1H),
8.80 (d, J ) 6.1 Hz, 1H), 9.44 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-o-tolylp ip er a zin e (50). Following gen-
eral procedure B, this product was obtained as a yellow oil
(yield 72%): [R] ) -47.0, c ) 1.27% in CHCl3. 1H NMR (CDCl3)
δ: 1.38 (s, 9H), 2.28 (s, 3H), 2.74 (m, 6H), 2.86 (s, 3H), 3.65
(m, 4H), 5.26 (t, J ) 7.2 Hz, 1H), 6.81 (dd, J ) 9.0 and 8.4 Hz,
3H), 7.02 (dd, J ) 8.9 and 8.5 Hz, 3H), 7.18 (t, J ) 7.4 Hz,
2H), 7.72 (t, J ) 7.7 Hz, 1H), 8.27 (dd, J ) 8.4 and 6.1 Hz,
2H), 8.54 (d, J ) 6 Hz, 1H), 8.82 (d, J ) 6 Hz, 1H), 9.43 (s,
1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(2-m eth oxyp h en yl)p ip er a zin e (51).
Following general procedure B, this product was obtained as
1
a yellow oil (yield 64%): [R] ) -61.0, c ) 1.54% in CHCl3. H
NMR (CDCl3) δ: 1.34 (s, 9H), 2.75 (s, 3H), 2.91 (m, 6H), 3.51
(m, 4H), 3.87 (s, 3H), 5.20 (t, J ) 7 Hz, 1H), 6.76 (d, J ) 8.5,
2H), 6.83 (t, J ) 6.6, 3H), 7.06 (d, J ) 8.5, 3H), 7.61 (t, J ) 6.6
Hz, 1H), 8.27 (m, 2H), 8.54 (d, J ) 6.0 Hz, 1H), 8.81 (d, J )
6.0 Hz, 1H), 9.42 (s, 1H).
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxyca r bon -
yltyr osyl]-4-o-tolylp ip er a zin e (60). Following general pro-
cedure B, this product was obtained as a white solid (yield
73%): mp 73-75 °C; [R] ) +22.5, c ) 1.3% in CH2Cl2. 1H NMR
(CDCl3) δ: 1.39 (s, 9H), 2.27 (s, 3H), 2.42 (m, 1H), 2.75 (m,
3H), 2.91 (d, J ) 6.7 Hz, 2H), 3.15 (m, 1H), 3.51 (m, 3H), 4.80
1-[(S)-O-Isoqu in olin esu lfon yl-N-ter t-bu tyloxycar bon yl-
N-m eth yltyr osyl]-4-(3-ch lor op h en yl)p ip er a zin e (52). Fol-
lowing general procedure B, this product was obtained as a
yellow oil (yield 98%): mp 79-81 °C; [R] ) -42.2, c ) 0.62%

1326 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Baraldi et al.
1
(m, 1H), 5.37 (d, J ) 8.5 Hz, 1H), 6.80 (d, J ) 8.5 Hz, 2H),
6.90 (d, J ) 7.8 Hz, 1H), 7.05 (t, J ) 8.4 Hz, 3H), 7.18 (t, J )
7.3 Hz, 2H), 7.54 (t, J ) 7.8, 1H), 8.22 (dd, J ) 8.2 and 7.3 Hz,
2H), 8.54 (d, J ) 6.2 Hz, 1H), 8.81 (d, J ) 6 Hz, 1H), 9.41 (s,
1H).
Gen er a l P r oced u r e for Rem ova l of th e Boc P r otectin g
Gr ou p fr om Com p ou n d s 35-60. The esters 35-60 (1.5
mmol) w er e stirred at room temperature in a mixture of TFA/
CH2Cl2 (1:1, 5 mL) for 3 h. The volatiles were removed in
vacuo, and the residue was diluted with 5% aqueous NaHCO3
(5 mL). The aqueous mixture was extracted with CH2Cl2
(3 × 5 mL), and the combined organic extracts were dried
(Na2SO4) and concentrated in vacuo. The residue obtained was
used for the next reaction without any purification.
Gen er a l P r oced u r e C for th e Syn th esis of Com p ou n d s
61-87. To a stirred solution of the appropriate free amine (0.5
mmol) in dry DCM (5 mL) were added Et3N (70 µL, 0.5 mmol,
1 equiv) and then dropwise isoquinolinesulfonyl chloride (1
mmol, 2 equiv) dissolved in DCM (3 mL), under cooling with
ice. The reaction mixture obtained was slowly warmed to room
temperature and stirred for 18 h. After this time, the mixture
was diluted with DCM (5 mL) and washed with saturated
aqueous NaHCO3 (2 mL), water (5 mL), and brine (5 mL). After
the layers were separated, the organic layer was dried
(Na2SO4) and concentrated in vacuo. The residue obtained,
subjected to purification by column chromatography using
a stepwise gradient of methanol (0.2-0.5%) in methylene
chloride, furnished the appropriate products 61-87.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-ben zylp ip er a zin e (61). Following general procedure C, this
product was obtained as a yellow solid: [R] ) -33.8, c ) 0.95%
in CHCl3. 1H NMR (CDCl3) δ: 1.95 (m, 1H), 2.16 (m, 1H), 2.25
(t, J ) 4.7 Hz, 2H), 2.49 (dd, J ) 12.4 and 4.6 Hz, 1H), 3.04
(m, 4H), 3.19 (dd, J ) 10.3 Hz, 2H), 3.40 (m, 4H), 5.06 (dd, J
) 10.3 and 4.5 Hz, 1H), 6.76 (d, J ) 8.6 Hz, 2H), 6.95 (d, J )
8.6 Hz, 2H), 7.27 (m, 5H), 7.59 (t, J ) 7.8 Hz, 1H), 7.68 (t, J
) 7.6 Hz, 1H), 8.25 (dd, J ) 8.2 and 7.7 Hz, 4H), 8.39 (d, J )
6.1 Hz, 1H), 8.56 (d, J ) 6.2 Hz, 1H), 8.66 (d, J ) 6.2 Hz, 1H),
8.83 (d, J ) 6.1 Hz, 1H), 9.35 (s, 1H), 9.43 (s, 1H). Anal.
(C39H37N5O6S2) C, H, N.
-46.0, c ) 0.73% in CHCl3. H NMR (CDCl3) δ: 2.42 (dd, J )
12.6 and 6.2 Hz, 1H), 2.63 (m, 1H), 2.99 (m, 5H), 3.22 (m, 2H),
3.55(m, 4H), 5.11 (dd, J ) 13.6 and 6 Hz, 1H), 6.63 (d, J ) 8.9
Hz, 2H), 6.77 (d, J ) 8.4 Hz, 2H), 6.96 (d, J ) 8.4 Hz, 2H),
7.54 (dd, J ) 8.8 and 7.8 Hz, 3H), 7.71 (t, J ) 7.8 Hz, 1H),
8.24 (d, J ) 7.6 Hz, 4H), 8.42 (d, J ) 6.3 Hz, 1H), 8.50 (d, J )
6.1 Hz, 1H), 8.68 (d, J ) 6.1 Hz, 1H), 8.81 (d, J ) 6.1 Hz, 1H),
9.36 (s, 1H), 9.41 (s, 1H). Anal. (C38H34N5O6S2I) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-p-tolylp ip er a zin e (66). Following the general procedure,
this product was obtained as a foamy yellow solid: [R] ) -41.5,
c ) 0.45% in CHCl3. 1H NMR (CDCl3) δ: 2.28 (s, 3H), 2.89 (m,
2H), 3.04 (s, 3H), 3.21 (m, 4H), 3.49 (m, 4H), 5.14 (dd, J ) 12
and 6.2 Hz, 1H), 6.77 (dd, J ) 6.2 and 3 Hz, 2H), 6.96 (d, J )
8.6 Hz, 2H), 7.09 (d, J ) 8.4 Hz, 2H), 7.49 (t, J ) 7.8 Hz, 1H),
7.70 (t, J ) 7.8 Hz, 1H), 8.13 (d, J ) 7.4 Hz, 2H), 8.17 (t, J )
8.8 Hz, 2H), 8.24 (t, J ) 7.8 Hz, 1H), 8.31 (d, J ) 7.2 Hz, 1H),
8.41 (d, J ) 6 Hz, 1H), 8.51 (d, J ) 6.2 Hz, 1H), 8.68 (d, J )
6.2 Hz, 1H), 8.81 (d, J ) 6 Hz, 1H), 9.35 (s, 1H), 9.40 (s, 1H).
Anal. (C39H37N5O6S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-m eth oxyp h en yl)p ip er a zin e (67). Following general
procedure C, this product was obtained as a white solid: [R]
1
) -48.4, c ) 0.94% in CHCl3. H NMR (CDCl3) δ: 2.48 (dd, J
) 12 and 4.4 Hz, 2H), 2.83 (m, 4H), 3.16 (s, 3H), 3.52 (m, 4H),
3.77 (s, 3H), 5.11 (dd, J ) 6.6 and 6.2 Hz, 1H), 6.76 (m, 6H),
6.96 (d, J ) 8.4 Hz, 2H), 7.51 (d, J ) 8 Hz, 1H), 7.69 (t, J ) 8
Hz, 1H), 8.23 (m, 4H), 8.41 (d, J ) 6.2 Hz, 1H), 8.51 (d, J )
6.2 Hz, 1H), 8.67 (d, J ) 6.2 Hz, 1H), 8.80 (d, J ) 6.2 Hz, 1H),
9.35 (s, 1H), 9.40 (s, 1H). Anal. (C39H37N5O7S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-n itr op h en yl)p ip er a zin e (68). Following general proce-
dure C, this product was obtained as a yellow solid: [R] ) -86,
c ) 1.02% in CHCl3. 1H NMR (CDCl3) δ: 2.34 (dd, J ) 14 and
6.2 Hz, 2H), 3.01 (s, 3H), 3.28 (m, 4H), 3.64 (m, 4H), 5.19 (dd,
J ) 13.8 and 6.2 Hz, 1H), 6.78 (d, J ) 5.2 Hz, 2H), 6.81 (d, J
) 5.6 Hz, 2H), 6.98 (d, J ) 8.6 Hz, 2H), 7.59 (t, J ) 7.8 Hz,
1H), 7.73 (t, J ) 7.8 Hz, 1H), 8.13 (d, J ) 7.4 Hz, 2H), 8.17 (d,
J ) 7.4 Hz, 2H), 8.20 (t, J ) 8.8 Hz, 1H), 8.23 (t, J ) 8.8 Hz,
1H), 8.44 (d, J ) 6.2 Hz, 1H), 8.50 (d, J ) 6.2 Hz, 1H), 8.68 (d,
J ) 6.2 Hz, 1H), 8.80 (d, J ) 6 Hz, 1H), 9.37 (s, 1H), 9.42 (s,
1H). Anal. (C38H34N6O8S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-p h en eth ylp ip er a zin e (62). Following general procedure C,
this product was obtained as a yellow solid: [R] ) +33.2, c )
1
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-cya n op h en yl)p ip er a zin e (69). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
0.84% in CH2Cl2. H NMR (CDCl3) δ: 1.94 (m, 6H), 2.47 (m,
2H), 2.73 (m, 5H), 3.04 (m, 4H), 4.31 (m, 1H), 6.12 (m, 1H),
6.63 (d, J ) 8.5 Hz, 2H), 6.86 (d, J ) 8.5 Hz, 2H), 7.22 (m,
5H), 7.62 (t, J ) 7.8 Hz, 2H), 8.23 (m, 5H), 8.51 (d, J ) 6 Hz,
1H), 8.68 (d, J ) 6.2 Hz, 1H), 8.80 (d, J ) 6.2 Hz, 1H), 9.32 (s,
1H), 9.41 (s, 1H). Anal. (C40H39N5O6S2) C, H, N.
1
-67.2, c ) 0.75% in CHCl3. H NMR (CDCl3) δ: 2.17 (dd, J )
12.6 and 6.2 Hz, 2H), 3.00 (s, 3H), 3.17 (m, 4H), 3.69 (m, 4H),
5.11 (dd, J ) 6.6 and 6 Hz, 1H), 6.80 (m, 4H), 6.97 (d, J ) 6.8
Hz, 2H), 7.52 (d, J ) 8.6 Hz, 1H), 7.58 (t, J ) 7.6 Hz, 1H),
7.72 (t, J ) 7.8 Hz, 1H), 8.30 (m, 4H), 8.41 (d, J ) 6 Hz, 1H),
8.49 (d, J ) 6 Hz, 1H), 8.67 (d, J ) 6.2 Hz, 1H), 8.81 (d, J )
6 Hz, 1H), 9.37 (s, 1H), 9.42 (s, 1H). Anal. (C39H34N6O6S2) C,
H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-flu or op h en yl)p ip er a zin e (63). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
1
-56.6, c ) 0.45% in CHCl3. H NMR (CDCl3) δ: 2.42 (dd, J )
12.6 and 6.2 Hz, 1H), 2.63 (m, 1H), 2.89 (m, 2H), 3.03 (s, 3H),
3.25 (dd, J ) 12.6 Hz, 2H) 3.55 (m, 4H), 5.12 (dd, J ) 12 and
6.2 Hz, 1H), 6.80 (m, 4H), 6.99 (t, J ) 8.5 Hz, 4H), 7.55 (t, J
) 7.9 Hz, 1H), 7.71 (t, J ) 7.9 Hz, 1H), 8.25 (d, J ) 8 Hz, 4H),
8.42 (d, J ) 6.3 Hz, 1H), 8.52 (d, J ) 6.3 Hz, 1H), 8.68 (d, J )
6.3 Hz, 1H), 8.81 (d, J ) 6.3 Hz, 1H), 9.36 (s, 1H), 9.41 (s,
1H). Anal. (C38H34N5O6S2F) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-a cetylp h en yl)p ip er a zin e (70). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
1
-56.9, c ) 1.12% in CHCl3. H NMR (CDCl3) δ: 2.42 (dd, J )
12.6 and 6.2 Hz, 1H), 2.53 (s, 3H), 2.90 (m, 1H), 3.02 (s, 3H),
3.21 (m, 5H), 3.63 (m, 3H), 5.14 (dd, J ) 12 and 6.2 Hz, 1H),
6.79 (t, J ) 8.5, 4H), 6.95 (d, J ) 8.5 Hz, 2H), 7.52 (t, J ) 7.8
Hz, 1H), 7.71 (t, J ) 7.8 Hz, 1H), 7.89 (d, J ) 8.9 Hz, 2H),
8.24 (d, J ) 7.4 Hz, 4H), 8.41 (d, J ) 6.1 Hz, 1H), 8.49 (d, J )
6.2 Hz, 1H), 8.68 (d, J ) 6.2 Hz, 1H), 8.79 (d, J ) 6.1 Hz, 1H),
9.35 (s, 1H), 9.39 (s, 1H). Anal. (C40H37N5O7S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-ch lor op h en yl)p ip er a zin e (64). Following general pro-
cedure C, this product was obtained as a yellow oil (yield
1
61%): [R] ) -43.4, c ) 0.89% in CHCl3. H NMR (CDCl3) δ:
2.44 (dd, J ) 12.6 and 6.2 Hz, 2H), 2.92 (m, 4H), 3.00 (s, 3H),
3.57 (m, 4H), 5.11 (dd, J ) 6.6 and 6.2 Hz, 1H), 6.76 (d, J )
8.6 Hz, 4H), 6.95 (d, J ) 8.6 Hz, 2H), 7.23 (d, J ) 6.6 Hz, 1H),
7.54 (d, J ) 8 Hz, 1H), 7.70 (t, J ) 7.8 Hz, 1H), 8.23 (m, 4H),
8.41 (d, J ) 6 Hz, 1H), 8.49 (d, J ) 6 Hz, 1H), 8.67 (d, J ) 6.2
Hz, 1H), 8.80 (d, J ) 6 Hz, 1H), 9.35 (s, 1H), 9.41 (s, 1H). Anal.
(C38H34N5O6S2Cl) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-flu or oben zyl)p ip er a zin e (71). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
1
-21.5, c ) 0.72% in CHCl3. H NMR (CDCl3) δ: 1.95 (m, 1H),
2.24 (m, 3H), 2.49 (dd, J ) 12.4 and 4.6 Hz, 1H), 3.04 (m, 4H),
3.14 (m, 2H), 3.38 (m, 4H), 5.06 (dd, J ) 10.3 and 4.5 Hz, 1H),
6.78 (d, J ) 8.4 Hz, 2H), 6.99 (d, J ) 8.4 Hz, 4H), 7.22 (m,
2H), 7.66 (t, J ) 7.8 Hz, 2H), 8.25 (m, 4H), 8.39 (d, J ) 6.2
Hz, 1H), 8.56 (d, J ) 6 Hz, 1H), 8.66 (d, J ) 6.3 Hz, 1H), 8.83
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-iod op h en yl)p ip er a zin e (65). Following general proce-
dure C, this product was obtained as a yellow solid: [R] )

Analogues of KN-62
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8 1327
(d, J ) 6.2 Hz, 1H), 9.36 (s, 1H), 9.44 (s, 1H). Anal.
(C39H36N5O6S2F) C, H, N.
7.54 (t, J ) 8 Hz, 1H), 7.72 (t, J ) 8 Hz, 1H), 8.26 (m, 4H),
8.42 (d, J ) 6.2 Hz, 1H), 8.51 (d, J ) 6.2 Hz, 1H), 8.68 (d, J )
6.2 Hz, 1H), 8.81 (d, J ) 6.2 Hz, 1H), 9.37 (s, 1H), 9.42 (s,
1H). Anal. (C38H34N5O6S2Cl) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-flu or oben zoyl)p ip er a zin e (72). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
-37.8, c ) 0.82% in CH2Cl2. 1H NMR (CDCl3) δ: 2.49 (m, 1H),
2.79 (m, 1H), 3.03 (s, 3H), 3.28 (m, 6H), 3.57 (m, 2H), 5.07
(dd, J ) 10.9 and 4.3 Hz, 1H), 6.79 (d, J ) 8.4 Hz, 2H), 6.97
(d, J ) 8.5 Hz, 2H), 7.12 (t, J ) 8.6 Hz, 2H), 7.44 (m, 2H),
7.72 (t, J ) 7.9 Hz, 2H), 8.31 (m, 5H), 8.54 (d, J ) 5.9 Hz,
1H), 8.68 (d, J ) 6 Hz, 1H), 8.84 (d, J ) 6 Hz, 1H), 9.38 (s,
1H), 9.45 (s, 1H). Anal. (C39H34N5O7S2F) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(3-tr iflu or om eth ylph en yl)piper azin e (79). Following gen-
eral procedure C, this product was obtained as a yellow solid:
[R] ) -61.5, c ) 0.4% in CHCl3. 1H NMR (CDCl3) δ: 2.48 (dd,
J ) 12 and 4.4 Hz, 1H), 2.75 (m, 1H), 2.93 (m, 5H), 3.30 (m,
2H), 3.62 (m, 4H), 5.11 (dd, J ) 6.6 and 6.2 Hz, 1H), 6.78 (d,
J ) 8.6 Hz, 2H), 6.99 (d, J ) 8.6 Hz, 4H), 7.15 (d, J ) 7.8 Hz,
1H), 7.40 (t, J ) 8.8 Hz, 1H), 7.55 (t, J ) 7.8 Hz, 1H), 7.72 (t,
J ) 7.7 Hz, 1H), 8.23 (m, 4H), 8.43 (d, J ) 6 Hz, 1H), 8.51 (d,
J ) 6.3 Hz, 1H), 8.68 (d, J ) 6.4 Hz 1H), 8.80 (d, J ) 6 Hz
1H), 9.36 (s, 1H), 9.41 (s, 1H). Anal. (C39H34N5O6S2F3) C, H,
N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-n itr oben zyl)p ip er a zin e (73). Following general proce-
dure C, this product was obtained as a yellow solid: [R] )
1
-28.4, c ) 0.37% in CHCl3. H NMR (CDCl3) δ: 1.95 (m, 1H),
2.27 (m, 3H), 2.49 (dd, J ) 12.4 and 4.6 Hz, 1H), 2.02 (m, 4H),
3.21 (dd, J ) 10.3 Hz, 2H), 3.49 (m, 4H), 5.12 (dd, J ) 10.3
and 4.5 Hz, 1H), 6.82 (d, J ) 8.4 Hz, 2H), 6.97 (d, J ) 8.6 Hz,
2H), 7.48 (d, J ) 8.4 Hz, 2H), 7.68 (d, J ) 8.3 Hz, 2H), 8.18 (d,
J ) 8.6 Hz, 3H), 8.30 (d, J ) 7.5 Hz, 3H), 8.40 (d, J ) 6 Hz,
1H), 8.55 (d, J ) 6.2 Hz, 1H), 8.67 (d, J ) 6 Hz, 1H), 8.83 (d,
J ) 6.3 Hz, 1H), 9.37 (s, 1H), 9.45 (s, 1H). Anal. (C39H36N6O8S2)
C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(o,m -xylyl)p ip er a zin e (80). Following general procedure
C, this product was obtained as a brown solid: [R] ) -17.5, c
1
) 1.91% in CH2Cl2. H NMR (CDCl3) δ: 2.19 (s, 3H), 2.27 (s,
3H), 2.49 (dd, J ) 12.4 and 4.6 Hz, 1H), 2.68 (m, 3H), 3.06 (s,
3H), 3.25 (m, 2H), 3.58 (m, 4H), 5.12 (dd, J ) 10.3 and 4.5 Hz,
1H), 6.76 (t, J ) 8.7 Hz, 2H), 6.84 (d, J ) 7.2 Hz, 1H), 7.03 (t,
J ) 8.7 Hz, 2H), 7.12 (t, J ) 8.7 Hz, 2H), 7.56 (t, J ) 7.7 Hz,
1H), 7.71 (t, J ) 7.7 Hz, 1H), 8.26 (m, 4H), 8.44 (d, J ) 6.1
Hz, 1H), 8.54 (d, J ) 6.2 Hz, 1H), 8.68 (d, J ) 6.2 Hz, 1H),
8.82 (d, J ) 6 Hz, 1H), 9.36 (s, 1H), 9.42 (s, 1H). Anal.
(C40H39N5O6S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(1-flu or op h en yl)p ip er a zin e (74). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
1
-58.4, c ) 0.44% in CHCl3. H NMR (CDCl3) δ: 2.49 (m, 2H),
3.06 (s, 3H), 3.22 (m, 4H), 3.63 (m, 4H), 5.11 (dd, J ) 13.6 and
6 Hz, 1H), 6.79 (m, 4H), 7.00 (m, 4H), 7.57 (t, J ) 8.2 Hz, 1H),
7.70 (t, J ) 7.8 Hz, 1H), 8.25 (m, 4H), 8.41 (d, J ) 6.2 Hz,
1H), 8.52 (d, J ) 6.2 Hz, 1H), 8.67 (d, J ) 6.2 Hz, 1H), 8.81 (d,
J ) 6.2 Hz, 1H), 9.36 (s, 1H), 9.42 (s, 1H). Anal. (C38H34N5O6S2F)
C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osy]l-
4-(3,4-d ich lor op h en yl)p ip er a zin e (81). Following general
procedure C, this product was obtained as a yellow solid: [R]
) -73.4, c ) 0.44% in CHCl3. 1H NMR (CDCl3) δ: 2.96 (m,
2H), 3.02 (s, 3H), 3.41 (m, 4H), 3.63 (m, 4H), 5.16 (dd, J )
13.6 and 6 Hz, 1H), 6.80 (m, 4H), 6.94 (t, J ) 8.4 Hz, 3H),
7.57 (t, J ) 8 Hz, 1H), 7.72 (t, J ) 8 Hz, 1H), 8.22 (m, 4H),
8.44 (d, J ) 6 Hz, 1H), 8.52 (d, J ) 6 Hz, 1H), 8.66 (d, J ) 6
Hz, 1H), 8.81 (d, J ) 6 Hz, 1H), 9.37 (s, 1H), 9.42 (s, 1H). Anal.
(C38H33N5O6S2Cl2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(2-ch lor op h en yl)p ip er a zin e (75). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
1
-39.2, c ) 0.92% in CHCl3. H NMR (CDCl3) δ: 2.48 (dd, J )
12.4 and 4.6 Hz, 2H), 2.83 (m, 2H), 3.07 (s, 3H), 3.23 (t, J )
10.4 Hz, 2H), 3.58 (m, 4H), 5.12 (dd, J ) 10.3 and 4.5 Hz, 1H),
6.77 (d, J ) 8.6 Hz, 2H), 6.97 (d, J ) 8.6 Hz, 4H), 7.23 (d, J )
7.8 Hz, 1H), 7.38 (d, J ) 8.8 Hz, 1H), 7.58 (t, J ) 7.8 Hz, 1H),
7.71 (t, J ) 7.7 Hz, 1H), 8.25 (dd, J ) 8.3 and 7.4 Hz, 4H),
8.42 (d, J ) 6.2 Hz, 1H), 8.54 (d, J ) 6 Hz, 1H), 8.68 (d, J )
5.9 Hz, 1H), 8.82 (d, J ) 6.2 Hz, 1H), 9.37 (s, 1H), 9.43 (s,
1H). Anal. (C38H34N5O6S2Cl) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-p yr id in -2-ylp ip er a zin e (82). Following general procedure
C, this product was obtained as a yellow solid: [R] ) -55.8, c
) 0.41% in CH2Cl2. 1H NMR (CDCl3) δ: 2.46 (dd, J ) 12.7
and 4.3 Hz, 1H), 2.91 (m, 1H), 3.05 (s, 3H), 3.21 (t, J ) 10.6
Hz, 3H), 3.42 (m, 3H), 5.09 (dd, J ) 10.4 and 4.4 Hz, 1H), 6.60
(d, J ) 8.6 Hz, 1H), 6.70 (d, J ) 8.4 Hz, 3H), 6.96 (d, J ) 8.4
Hz, 2H), 7.53 (m, 2H), 7.70 (t, J ) 7.8 Hz, 1H), 8.12 (d, J )
7.3 Hz, 1H), 8.22 (m, 3H), 8.30 (d, J ) 7.3 Hz, 1H), 8.41 (d, J
) 6.2 Hz, 1H), 8.51 (d, J ) 6.2 Hz, 1H), 8.67 (d, J ) 6.3 Hz,
1H), 8.80 (d, J ) 6.1 Hz, 1H), 9.35 (s, 1H), 9.40 (s, 1H). Anal.
(C37H34N6O6S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-o-tolylp ip er a zin e (76). Following general procedure C, this
product was obtained as a yellow solid: [R] ) -39.2, c ) 0.92%
1
in CHCl3. H NMR (CDCl3) δ: 2.28 (s, 3H), 2.35 (m, 1H), 2.49
(dd, J ) 12.4 and 4.6 Hz, 1H), 2.68 (m, 2H), 3.06 (s, 3H), 3.30
(m, 2H), 3.58 (m, 4H), 5.12 (dd, J ) 10.3 and 4.5 Hz, 1H), 6.81
(t, J ) 9.0 and 8.4 Hz, 3H), 7.02 (dd, J ) 8.9 and 8.5 Hz, 3H),
7.18 (t, J ) 7.4 Hz, 2H), 7.56 (t, J ) 7.7 Hz, 1H), 7.72 (t, J )
7.7 Hz, 1H), 8.27 (dt, J ) 8.4 and 6.1 Hz, 4H), 8.42 (d, J ) 6.2
Hz, 1H), 8.54 (d, J ) 6 Hz, 1H), 8.68 (d, J ) 6.2 Hz, 1H), 8.82
(d, J ) 6 Hz, 1H), 9.37 (s, 1H), 9.43 (s, 1H). Anal. (C39H37N5O6S2)
C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-p yr im id in -2-ylp ip er a zin e (83). Following general proce-
dure C, this product was obtained as a yellow solid: [R] )
-54.6, c ) 0.76% in CH2Cl2. 1H NMR (CDCl3) δ: 2.46 (dd, J )
12.7 and 4.3 Hz, 1H), 3.00 (m, 1H), 3.06 (s, 3H), 3.41 (m, 6H),
3.80 (m, 2H), 5.09 (dd, J ) 10.4 and 4.4 Hz, 1H), 6.56 (t, J )
8.6 Hz, 1H), 6.74 (d, J ) 8.5 Hz, 2H), 6.96 (d, J ) 8.5 Hz, 2H),
7.57 (t, J ) 7.8 Hz, 1H), 7.70 (t, J ) 7.8 Hz, 1H), 8.21 (t, J )
7.6 Hz, 4H), 8.33 (m, 2H), 8.40 (d, J ) 6.3 Hz, 1H), 8.52 (d, J
) 6.1 Hz, 1H), 8.67 (d, J ) 6 Hz, 1H), 8.82 (d, J ) 6 Hz, 1H),
9.35 (s, 1H), 9.41 (s, 1H). Anal. (C36H33N7O6S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-ben zylp ip er id in e (84). Following general procedure C, this
product was obtained as a yellow solid: [R] ) -24, c ) 0.42%
in CH2Cl2. 1H NMR (CDCl3) δ: 1.50 (m, 2H), 2.39 (m, 6H),
2.70 (m, 1H), 3.04 (s, 3H), 3.25 (m, 2H), 3.80 (m, 1H), 4.40 (m,
1H), 5.10 (m, 1H), 6.73 (m, 1H), 6.88 (d, J ) 8.6 Hz, 2H), 7.05
(m, 3H), 7.24 (m, 3H), 7.68 (m, 2H), 8.26 (m, 4H), 8.41 (d, J )
6.1 Hz, 1H), 8.56 (d, J ) 6.2 Hz, 1H), 8.68 (d, J ) 6.2 Hz, 1H),
8.82 (d, J ) 6 Hz, 1H), 9.35 (s, 1H), 9.43 (s, 1H). Anal.
(C40H38N4O6S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osy]-
4-(2-m eth oxyp h en yl)p ip er a zin e (77). Following general
procedure C, this product was obtained as a yellow solid: [R]
1
) -29.8, c ) 0.45% in CHCl3. H NMR (CDCl3) δ: 2.51 (dd, J
) 12.6 and 6.2 Hz, 1H), 2.62 (m, 1H), 2.87 (m, 2H), 3.07 (s,
3H), 3.22 (m, 2H), 3.56 (m, 4H), 3.87 (s, 3H), 5.15 (dd, J )
13.6 and 6 Hz, 1H), 6.76 (d, J ) 8.6 Hz, 2H), 6.97 (d, J ) 7.9
Hz, 4H), 7.30 (m, 1H), 7.63 (m, 3H), 8.11 (d, J ) 8.1 Hz, 1H),
8.27 (d, J ) 7.3 Hz, 3H), 8.44 (m, 1H), 8.54 (d, J ) 6 Hz, 1H),
8.75 (m, 2H), 9.40 (m, 2H). Anal. (C39H37N5O7S2) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(3-ch lor op h en yl)p ip er a zin e (78). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
1
-58.8, c ) 0.6% in CHCl3. H NMR (CDCl3) δ: 2.42 (m, 2H),
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)tyr osyl]-4-(4-flu o-
r op h en yl)p ip er a zin e (85). Following general procedure C,
this product was obtained as a yellow solid: [R] ) +47.7, c )
2.99 (s, 3H), 3.22 (m, 4H), 3.59 (m, 4H), 5.12 (dd, J ) 12 and
6.2 Hz, 1H), 6.79 (m, 4H), 6.89 (m, 3H), 7.21 (t, J ) 8 Hz, 1H),

1328 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 8
Baraldi et al.
1
0.62% in CH2Cl2. H NMR (CDCl3) δ: 2.22 (m, 2H), 2.50 (m,
cellular Ca2+ concentration was determined with the 340/380
excitation ratio at an emission wavelength of 500 nM. All
experiments were repeated three times.
Cytok in e Relea se. IL-1â release was measured in macro-
phage monolayers primed for 2 h with bacterial endotoxin
(lipopolysaccharide, LPS) at 10 µg/mL and was stimulated with
3 mM ATP for 30 min. Inhibitors, when used, were added 5
min prior to ATP. Supernatants were centrifuged for 5 min at
900g to remove floating cells and were assayed for IL-1â
content by ELISA (R&D Systems, Minneapolis, MN).
4H), 3.06 (m, 3H), 3.25 (m, 1H), 4.35 (m, 1H), 5.99 (d, 1H),
6.65 (d, J ) 8.4 Hz, 2H), 6.78 (m, 2H), 6.88 (d, J ) 8.5 Hz,
2H), 6.99 (t, J ) 8.5 Hz, 2H), 7.58 (m, 2H), 8.16 (t, J ) 8 Hz,
2H), 8.27 (t, J ) 6.5 Hz, 3H), 8.51 (d, J ) 6.3 Hz, 1H), 8.70 (d,
J ) 6.3 Hz, 1H), 8.82 (d, J ) 6 Hz, 1H), 9.30 (s, 1H), 9.41 (s,
1H). Anal. (C37H32N5O6S2F) C, H, N.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)tyr osyl]-4-o-tolyl-
p ip er a zin e (86). Following general procedure C, this product
was obtained as a yellow solid: [R] ) +80.3, c ) 1% in CH2-
Cl2. 1H NMR (CDCl3) δ: 2.24 (s, 3H), 2.36 (m, 3H), 2.54 (m,
1H), 2.80 (d, J ) 7.2 Hz, 2H), 2.93 (m, 1H), 3.06 (m, 1H), 3.25
(m, 2H), 4.35 (t, J ) 7.5 Hz, 1H), 6.09 (d, J ) 9.1 Hz, 1H), 6.4
(d, J ) 8.3 Hz, 2H), 6.85 (dd, J ) 8.6 and 7.8 Hz, 3H), 7.03 (t,
J ) 7.4 Hz, 1H), 7.18 (m, 2H), 7.55 (t, J ) 7.9 Hz, 1H), 7.63 (t,
J ) 7.7 Hz, 1H), 8.24 (d, J ) 7.5 Hz, 5H), 8.52 (d, J ) 6 Hz,
1H), 8.69 (d, J ) 6.2 Hz, 1H), 8.82 (d, J ) 6 Hz, 2H), 9.33 (s,
1H), 9.41 (s, 1H). Anal. (C38H35N5O6S2) C, H, N.
Ack n ow led gm en t. We thank the Italian Ministry
for Education and Scientific Research (MIUR) (ex 40%),
the National Research Council of Italy (Target Project
on Biotechnology), the Italian Association for Cancer
Research, the Italian Space Agency, and University of
Ferrara for generous financial support of this work. We
also thank Professor Ernesto Damiani (University of
Padova) for help in the measurement of CaMII kinase
activity. Maria del Carmen Nunez is the recipient of a
Fundacio`n Ramo`n Areces fellowship.
1-[(S)-N,O-Bis(isoqu in olin esu lfon yl)-N-m eth yltyr osyl]-
4-(4-a m in op h en yl)p ip er a zin e (87). Following general pro-
cedure C, this product was obtained as a yellow solid: [R] )
1
-48.4, c ) 0.90% in CHCl3. H NMR (CDCl3) δ: 2.48 (m, 2H),
2.83 (m, 4H), 3.16 (s, 3H), 3.52 (m, 4H), 4.00 (m, 2H), 5.11
(dd, J ) 6.6 and 6.2 Hz, 1H), 6.28 (d, J ) 8.3 Hz, 2H), 6.34 (d,
J ) 8.3 Hz, 2H), 6.76 (d, J ) 8.4 Hz, 2H), 6.96 (d, J ) 8.4 Hz,
2H), 7.51 (d, J ) 8 Hz, 1H), 7.69 (t, J ) 8 Hz, 1H), 8.23 (m,
4H), 8.41 (d, J ) 6.2 Hz, 1H), 8.51 (d, J ) 6.2 Hz, 1H), 8.67 (d,
J ) 6.2 Hz, 1H), 8.80 (d, J ) 6.2 Hz, 1H), 9.35 (s, 1H), 9.40 (s,
1H). Anal. (C38H36N6O6S2) C, H, N.
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