Carbonic anhydrase activators: Amino acyl/dipeptidyl histamine derivatives bind with high affinity to isozymes I, II and IV and act as efficient activators

Article abstract of DOI:10.1016/S0968-0896(99)00227-8

Reaction of histamine (Hst) with tetrabromophthalic anhydride and protection of its imidazole moiety with tritylsulfenyl chloride, followed by hydrazinolysis, afforded N-1-tritylsulfenyl-histamine, a key intermediate which was further derivatized at its aminoethyl moiety. Reaction of the key intermediate with N-Boc-amino acids/dipeptides (Boc-AA) in the presence of carbodiimides afforded, after deprotection of the imidazolic and amino moieties, a series of compounds with the general formula AA-Hst (AA=amino acyl; dipeptidyl). The new derivatives were assayed as activators of three carbonic anhydrase (CA) isozymes, hCA I, hCA II (cytosolic forms) and bCA IV (membrane-bound form). Efficient activation was observed against all three isozymes, but especially against hCA I and bCA IV, with affinities in the nanomolar range for the best compounds. hCA II was, on the other hand, activatable with affinities around 10-20 nM. This new class of CA activators might lead to the development of drugs/diagnostic agents for the CA deficiency syndrome, a genetic disease of bone, brain and kidneys. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00227-8

Bioorganic & Medicinal Chemistry 7 (1999) 2915±2923
Carbonic Anhydrase Activators: Amino Acyl/Dipeptidyl
Histamine Derivatives Bind with High Anity to Isozymes
I, II and IV and Act as Ecient Activatorsy
Claudiu T. Supuran* and Andrea Scozzafava
Â
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Florence, Italy
Received 31 March 1999; accepted 21 July 1999
AbstractÐReaction of histamine (Hst) with tetrabromophthalic anhydride and protection of its imidazole moiety with tritylsulfenyl
chloride, followed by hydrazinolysis, a€orded N-1-tritylsulfenyl-histamine, a key intermediate which was further derivatized at its
aminoethyl moiety. Reaction of the key intermediate with N-Boc-amino acids/dipeptides (Boc-AA) in the presence of carbodiimides
a€orded, after deprotection of the imidazolic and amino moieties, a series of compounds with the general formula AA-Hst
(AA=amino acyl; dipeptidyl). The new derivatives were assayed as activators of three carbonic anhydrase (CA) isozymes, hCA I,
hCA II (cytosolic forms) and bCA IV (membrane-bound form). Ecient activation was observed against all three isozymes, but
especially against hCA I and bCA IV, with anities in the nanomolar range for the best compounds. hCA II was, on the other
hand, activatable with anities around 10±20 nM. This new class of CA activators might lead to the development of drugs/diag-
nostic agents for the CA de®ciency syndrome, a genetic disease of bone, brain and kidneys. # 1999 Elsevier Science Ltd. All rights
reserved.
Introduction
syndrome, a genetic disease of bone, brain and kidney
a€ecting a large number of patients.15 In this condition,
a certain CA isozyme gene (generally CA II, I or IV) is
either not expressed, or its protein product is unstable
due to deleterious mutations, and the corresponding CA
isozyme is absent in the blood, kidney or lung of such
patients. No pharmacologically speci®c treatment for
this condition is available up to now. CA activators are
also important for understanding the CA catalytic and
inhibition mechanisms.8±12
Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors of the
unsubstituted sulfonamide type, RSO2NH2, are widely
used drugs for the treatment or prevention of a variety
of diseases, such as: glaucoma,2,3 epilepsy,4 gastric and
duodenal ulcers,5 or acid±base disequilibria.6 In con-
trast to inhibitors, activators of this enzyme (for which
at least 14 di€erent isozymes have been isolated up to
now in higher vertebrates)7 were much less investigated.
Only recently the X-ray crystallographic structures of
the ®rst adducts of the physiologically relevant isozyme
II (hCA II) with the activators histamine (Hst)8 and
phenylalanine (in this case a tertiary complex, in which
azide is also bound to the Zn(II) ion)9 have been repor-
ted by this group. Furthermore, few other quantitative
structure±activity relationship (QSAR)10±12 or synthetic
chemistry13,14 studies have been reported in the ®eld of
CA activators, although some of these compounds
might be used in the treatment of the CA de®ciency
The lead molecule considered by us for obtaining tighter
binding CA activators was histamine 1 itself. As seen
from the X-ray coordinates with which Figure 1 was
generated, the activator molecule is bound at the
entrance of the hCA II active site cavity, where it is
anchored by hydrogen bonds to amino acid side-chains
and to water molecules. Such hydrogen bonds involve
only the nitrogen atoms of the imidazole moiety,
whereas the terminal aliphatic amino group is not
experiencing any contact with the enzyme, but is
extending away from the cavity into the solvent. On the
other hand, the Nd1 and Ne2 atoms of the histidine
imidazole ring are engaged in hydrogen bonds with the
side-chains of Asn 62, His 64, Gln 92 and with Wat152.8
Thus, it appeared of interest to derivatize the lead at its
Key words: Carbonic anhydrase; histamine; amino acid; dipeptide;
enzyme activators; proton shuttle.
* Corresponding author. Tel.: +39-055-275-7552; fax: +39-055-275-
7555; e-mail: cts@bio.chim.uni®.it
y See ref 1.
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00227-8

2916
C. T. Supuran, A. Scozzafava / Bioorg. Med. Chem. 7 (1999) 2915±2923
Results
Synthesis
Compounds reported in the present work were prepared
starting from N-1-tritylsulfenyl-histamine 3 (obtained as
shown in Scheme 1) and Boc-protected amino acids/
dipeptides, in the presence of carbodiimide derivatives.
Treatment of the intermediates 4 with TFA, and
removal of the tritylsulfenyl moiety with HCl±dioxane,
a€orded the new compounds A1±A24, of the type AA-
Hst or AA1-AA2-Hst, respectively.
CA activation study
Activation of three CA isozymes, hCA I, hCA II and
bCA IV (h=human; b=bovine isozymes), with Hst and
the new compounds A1±A24 is shown in Table 1.
Detailed activation curves with Hst and some of the
new derivatives are shown in Figure 2.
Figure 1. hCA II±histamine adduct: the Zn(II) ion (central sphere)
and its three histidine ligands (His 94, His 96 and His 119) are shown
at the center of the active site, whereas histamine (numbered as Hst
264) is situated at the entrance in it, between residues His 64 and Gln
92. The ®gure was generated from the X-ray coordinates of the hCA
II±histamine adduct reported by this group,8 with the program Ras-
Mol for Windows 2.6. The coordinates of this structure are deposited
in the Brookhaven Protein Database (PDB entry 4TST).
Discussion
The study of CA activators has been overlooked since a
report of hCA II activation by His,21 followed by its
subsequent retraction,22 and consideration that the
activation initially observed was due to chelation of
trace Cu(II) ions (which inhibit CA), eventually present
in the enzyme preparation, by EDTA added in the buf-
fer. Obviously, the experimental protocols of Silver-
man's laboratory presented many unresolved problems,
as explained by us in a previous work,23 and this issue of
CA activation has not thereafter been re-investigated by
other researchers. Only recently, by the report of the
®rst X-ray crystallographic data8,9 of adducts of Hst
and phenylalanine with hCA II, has this topic again
received deserved attention, and some types of high
anity activators investigated mainly by this8±19 and
Chegwidden's groups.24
aliphatic NH2 moiety, just in order to exploit the energy
of binding of such modi®ed groups with amino acid
residues at the edge of the active site. This approach has
been successfully used both by Whitesides'16,17 and our
groups8,18,19 for the design of tight-binding, isozyme-
speci®c sulfonamide CA inhibitors. Moreover, recently
we1 have reported some tosylureido-amino acyl deriva-
tives of histamine (at the aliphatic amino group) pos-
sessing high anities for the three CA isozymes
mentioned above. Thus, it appeared of interest to
explore other types of moieties that might be attached at
the aliphatic end of the molecule, and amino acyl
groups appeared of great interest, due to the possible
interactions of these highly polarized moieties with
amino acid residues at the edge of the active site (which
presumably would lead to increased stabilities of the
enzyme±activator adducts), as well as the presence of a
second proton shuttle moiety (the free amino group)
which might in¯uence positively the rate-limiting step
of the catalytic cycle, which is a proton transfer
process from the active site cavity to the reaction
medium.8,9
In this paper, we report the synthesis of a series of
amino acyl/dipeptidyl histamine derivatives possessing
the general formula AA-Hst, obtained by reaction of
appropriately protected1,20 Hst with Boc-protected
amino acids/dipeptides, in the presence of carbodi-
imides, followed by removal of the two protecting
groups. The new compounds were assayed as activators
of three CA isozymes, hCA I, hCA II and bCA IV
(h=human, b=bovine isozyme), and generally showed
very good activities. SAR in this series of derivatives is
also discussed.
Scheme 1. Reagents: (a) tetrabromophthalic anhydride; (b) tri-
tylsulfenyl chloride; (c) TFA; (d) 4 M HCl±dioxane.

C. T. Supuran, A. Scozzafava / Bioorg. Med. Chem. 7 (1999) 2915±2923
Table 1. CA isozymes I, II and IV activation with histamine (Hst) 1
2917
and the aminoacyl/dipeptidyl derivatives AA-Hst, A1±A24. The acti-
vation constant KA is de®ned similarly to the inhibition constant KI, as
the equilibrium constant for the reaction: E+A$E±A (where E,
enzyme; A, activator; E±A, enzyme±activator complex)8
a
No.
AA
KA (mM)
Yield
hCA Ib
hCA IIb
bCA IVc
1
A1
A2
A3
A4
A5
A6
A7
Ð
Gly
l-Ala
2
125
10
11
9
7
7
6
7
7
6
41
1.5
2.3
1.2
1.0
1.1
5.1
4.6
4.1
4.0
2.1
0.3
0.3
0.2
2.3
2.2
4.1
3.1
2.1
2.1
1.2
0.01
0.003
0.005
0.008
Ð
54
50
60
67
45
62
39
55
78
80
53
61
70
84
83
95
66
42
49
51
35
61
39
30
0.21
0.20
0.20
0.15
0.13
0.18
0.18
0.20
0.16
0.14
0.02
0.03
0.01
0.07
0.06
0.20
0.16
0.12
0.13
0.11
0.001
0.001
0.003
0.004
b-Ala
Figure 2. hCA II activation (0.012 mM) with histamine (curve 1),
compound A11 (curve 2) and compound A22 (curve 3) in the con-
centration range of 1 nM±10 mM, for the hydrolysis of 4-nitrophenyl
acetate. Control CA activity in the absence of activator was taken as
100%. Substrate concentration was 2.5 mM, 10 mM Tris bu€er, pH
7.40, at 25ꢀC and ionic strength of 0.1 (K2SO4).
GABA
GlyGly
l-Val
l-Leu
l-Ile
l-Asn
l-Gln
l-Arg
l-Lys
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
5
0.3
0.4
0.4
7
5
10
8
6
5
5
The data of Table 1 show signi®cant di€erences between
the investigated isozymes in their behavior towards both
`classical' activators, such as Hst 1, as well as the new
class of activators synthesized in the present work.
Thus, Hst 1 is a potent hCA I activator, and a relatively
weak hCA II activator, whereas isozyme bCA IV pos-
sesses an intermediate behavior. The most interesting
®nding of the present study is represented by the high
susceptibility of the cytosolic isozyme, hCA II, to be
activated by some of the derivatives of Hst of types A1±
A24, as compared to the lead molecule (compounds
with activation constants in the 0.01±0.03 mM range
were frequently obtained). Moreover, the highly abun-
dant and most prone to activation (by Hst) isozyme
hCA I was also susceptible to activation by the new
derivatives reported here (with constants in the nano-
molar range for the most active derivatives), but di€er-
ences of activity are not so pronounced as compared to
the situation for the rapid isozyme hCA II. bCA IV, on
the other hand, had an intermediate behavior towards
the new class of activators, with activation constants in
the 0.008±0.02 mM range for the most active com-
pounds. Ecient CA activators were: (i) derivatives of
basic amino acids (Arg, Lys, His), such as A11±A13, as
well as the phenylglycine and phenylalanine derivatives
A14 and A15; (ii) slightly less active were the com-
pounds derived from Pro, Pip, Nip, Inp, Asn, Gln, as
well as the hydrophobic amino acid derivatives (Val,
Leu, Ile, Trp); (iii) GlyGly (A5) and GABA (A4) deri-
vatives were more active than the b-Ala derivative (A3),
which in turn was more active than the Ala or Gly
derivatives (A2 and A1); (iv) the best activators in this
series were those derived from dipeptides such as Gly-
His, b-Ala-His (carnosine), Phe-Pro or Pro-Gly. These
compounds possessed activation constants in the 1±
5 nM range against hCA I and bCA IV, and 10±30 nM
range against hCA II. Probably the many heteroatoms
present in these dipeptidyl moieties confer to the
obtained compounds a `sticky' nature, i.e. they are able
to participate in many interactions with amino acid
l-His
l-Phgd
l-Phe
l-Trp
l-Pro
l-Pipe
d,l-Nipf
d,l-Inpg
l-GlyHis
l-b-AlaHis
l-PhePro
l-ProGly
0.02
0.01
0.03
0.03
a
Mean from at least three determinations by the esterase method.34
Standard error was in the range of 5±10%.
Human cloned isozyme.
Puri®ed from bovine lung microsomes.33
Phg=phenylglycine.
Pip=pipecolic acid (piperidine-2-carboxylic acid).
Nip=nipecotic acid (piperidine-3-carboxylic acid).
Inp=isonipecotic acid (piperidine-4-carboxylic acid).
b
c
d
e
f
g
The key intermediate for obtaining novel types of acti-
vators reported in this paper, N-1-tritylsulfenyl-Hst 3,
was obtained by non-exceptional procedures, involving
the initial protection of the primary amine moiety by
means of phthalimide derivatives, followed by protec-
tion of the imidazolic NH moiety with tritylsulfenyl
chloride, and hydrazinolysis of the phthalimido moiety
in mild conditions (Scheme 1). The overall yield of the
three steps was good (around 80%) and the puri®cation
procedures quite simple. Subsequent reaction of the key
intermediate 3 with Boc-amino acid/dipeptide deriva-
tives21,25,26 in the presence of carbodiimides27 a€orded a
series of N-tritylsulfenylated compounds 4, which were
deprotected in standard conditions, leading to the
desired derivatives A1±A24. All the new compounds
1
reported here have been characterized by IR, H and
13C NMR spectroscopy, as well as elemental analysis
(‹0.4% of the theoretical data, calculated for the pro-
posed formulas).

2918
C. T. Supuran, A. Scozzafava / Bioorg. Med. Chem. 7 (1999) 2915±2923
residues from the active site, thus assuring the forma-
tion of very stable enzyme±activator (E±A) adducts.
range of 4.0±9.0 in their molecule, thus assuring alter-
native proton transfer pathways. Indeed, the active site
edges of all three CA isozymes investigated by us con-
tain a high proportion of polar amino acid residues
which might interfere with amino acyl/dipeptidyl groups
by means of a multitude of interaction types. In fact
such amino acid residues also explain the di€erent cat-
alytic properties of the diverse isozymes, as well as their
diverse susceptibility to be inhibited/activated by mod-
ulators of activity.7,8 For instance, the entrance of the
active site of isozyme hCA II contains a cluster of six
histidine residues (His 3, His 4, His 10, His 15, His 17
and His 64), some of which possess di€erent con-
formations (as shown by X-ray crystallography)8,9
which could easily participate in hydrogen bond for-
mation (as well as other types of interactions) with the
Hst derivatives reported here. This might in fact
explain the greater eciency of the compounds repor-
ted in the present work in activating this isozyme, as
compared to Hst, which is a relatively weak hCA II
activator, and might lead to the development of phar-
macological agents from this class of compounds.
Activators are also critical for understanding the CA
catalytic mechanism as well as for explaining the great
di€erences in activity between the many CA isozymes,8
some of which have an unknown physiological function
for the moment.
A special mention should also be made regarding com-
pounds such as A11, A12, A13, A21 or A22, that possess
multiple moieties able to shuttle protons, in addition to
the Hst imidazolic one (the a-, b-, or g-NH2, plus the
guanidino-, e-amino- or imidazolic ring NH moieties,
respectively), and behave as very e€ective CA activators
against all three isozymes investigated here.
Typical activation curves with some of the best activa-
tors reported here (such as A11, A22) and Hst as stan-
dard, for hCA II catalyzed 4-nitrophenyl acetate
hydrolysis, are shown in Figure 2. It is obvious from the
data shown in this ®gure that Hst starts to activate hCA
II signi®cantly only at concentrations higher than
50 mM, then reaching a plateau of around 180% of the
control activity at concentrations of 1 mM or higher. In
contrast, the new activators show a much more
pronounced activation at lower concentrations (0.01±
1 mM), the above-mentioned plateau is reached at
lower activator concentrations, and the level of hCA II
activity enhancement is much higher (220±260%) as
compared to the corresponding histamine data.
Experimental
Melting points (mp) were determined with a heating
plate microscope and are not corrected; IR spectra were
obtained in KBr pellets with a Perkin±Elmer 16PC FT-
1
Similarly to all CA activators reported up to now, the
compounds obtained in the present work presumably
intervene in the catalytic cycle of the enzyme, leading to
the formation of CA±activator complexes (similarly to
the enzyme±inhibitor adducts, but without substitution
of the metal bound solvent molecule), in which the
activator bound within the active site facilitates proton
transfer processes (which represent the rate-limiting step
in catalysis).8±19 The driving force of this e€ect is prob-
ably the fact that intramolecular reactions are more
rapid than intermolecular ones. Thus, in the presence of
activators (symbolized as `A'), the rate-limiting step in
the CA catalytic cycle is described by eq (1) below:8±19
IR spectrometer and H NMR spectra with a Varian
300CXP apparatus in solvents speci®ed in each case.
Chemical shifts are expressed as d values relative to
Me4Si as standard. Elemental analyses were done by
combustion for C, H, N with an automated Carlo Erba
analyzer, and were ‹0.4% of the theoretical values.
Preparative high performance liquid chromatography
(HPLC) was done on C18 reversed-phase m-Bondapack
or Dynamax-60A (25Â250 mm) columns with a Beck-
mann HPLC-3069 instrument.
Compounds used in synthesis (Hst, natural and non-
acids/dipeptides,
natural
Boc-protected
amino
tritylsulfenyl chloride, tetrabromophthalic anhydride,
hydrazine, TFA, etc.) were commercially available
compounds (from Sigma, Acros or Aldrich) or were
prepared by standard procedures (from Boc-On and the
corresponding amino acids/dipeptides). Acetonitrile,
acetone, dioxane (E. Merck) or other solvents used in
the synthesis were doubly distilled and kept on mole-
cular sieves in order to maintain them in anhydrous
conditions.
EZn2‡ OH2 ‡ A ()
Â
Ã
Â
Ã
‡
AH
EZn2‡ OH2 A () EZn2‡ HO
enzyme activator complexes
ꢀ1†
‡
() EZn2‡ OH ‡ AH :
Compounds of the types reported here possess the imi-
dazolic Hst moiety which can participate in the proton
transfer processes between the active site and the envir-
onment (similarly to the lead, Hst 1), but due to the
presence of the amino acyl/dipeptidyl tails also present
in their molecule, they can bind more e€ectively to the
enzyme, thus allowing for much more ecient activation
processes as compared to 1. Furthermore, these tails can
also participate in ecient proton transfer processes due
to the presence of other groups with pKa values in the
hCA I and hCA II cDNAs were expressed in Escher-
ichia coli strain BL21 (DE3) from the plasmids pACA/
hCA I and pACA/hCA II described by Forsman et al.28
(the two plasmids were a gift from Professor Sven
Lindskog, Umea University, Sweden). Cell growth con-
ditions were those described by Lindskog's group,29 and
enzymes were puri®ed by anity chromatography

C. T. Supuran, A. Scozzafava / Bioorg. Med. Chem. 7 (1999) 2915±2923
2919
according to the method of Khalifah et al.30 Enzyme
concentrations were determined spectrophotometrically
solution containing 3 was brought to pH 7 with solid
NaHCO3, brought to a small volume by in vacuo eva-
poration of the solvent, and the precipitated 3 was then
recrystallized from ethanol (yield of 80%, based on
Hst, after the three steps described above). Tan crys-
tals, mp 177±178ꢀC; 1H NMR (300 MHz, TFA), d,
ppm: 2.47 (t, 2H, J=7.0 Hz, CH2); 2.96 (q, 2H, J=6.2,
12.5 Hz, H2NCH2); 4.23 (m, 2H, NH2); 7.10±7.30 (m,
15H, trityl); 7.34 (m, 1H, imidazole CH); 8.35 (s, 1H,
imidazole CH). Anal. found: C, 75.12; H, 5.83; N,
10.88%; C24H23N3S requires: C, 74.77; H, 6.01; N,
10.90%.
1
at 280 nm, utilizing a molar absorptivity of 49 mM
1
1
1
cm
for hCA I and 54 mM
cm
for hCA II,
respectively, based on Mr ˆ 28:85 kDa for hCA I, and
29.30 kDa for hCA II, respectively.31,32 bCA IV was
isolated and puri®ed to homogeneity from bovine
lung microsomes as described by Maren et al. and its
concentration has been determined by titration with
ethoxzolamide.33
Initial rates of 4-nitrophenyl acetate hydrolysis cata-
lyzed by di€erent CA isozymes were monitored spec-
trophotometrically, at 400 nm, with a Cary 3 instrument
interfaced with an IBM compatible PC.34 Solutions of
General procedure for the preparation of compounds A1±
A24. An amount of 10 mM N-1-tritylsulfenyl-histamine
3 was dissolved in 50 mL of anhydrous acetonitrile and
then treated with a solution obtained from 10 mM of
N-Boc-amino acid/dipeptide (10 mM) dissolved in
10 mL of the same solvent, followed by 10 mM of liquid
substrate were prepared in anhydrous acetonitrile; the
2
substrate concentrations varied between 2Â10
and
1Â10 6 M, working at 25ꢀC. A molar absorption coef-
1
1
®cient e of 18,400 M cm was used for the 4-nitro-
phenolate formed by hydrolysis, in the conditions of the
experiments (pH 7.40), as reported in the literature.34
Non-enzymatic hydrolysis rates were always subtracted
from the observed rates. Duplicate experiments were
done for each activator concentration, and the values
reported throughout the paper are the mean of such
results. Stock solutions of activator (1 mM) were pre-
pared in distilled±deionized water with 10±20% (v/v)
DMSO and dilutions up to 0.01 nM were done there-
after with distilled±deionized water. Activator and
enzyme solutions were preincubated together for 10 min
at room temperature prior to assay, in order to allow
for the formation of the E±A complex. The activator
constant KA was determined as described in ref 8. Con-
trol experiments done in the presence of 10 mM EDTA
added in the assay bu€er did not in¯uence in any way
the activation data reported in this paper, proving once
again that the CA activation is not due to chelation of
adventitious metal ions possibly present in the enzyme
preparations. Enzyme concentrations were 3.2 nM for
hCA II, 9 nM for hCA I and 16 nM for bCA IV (this
isozyme has a decreased esterase activity35 and higher
concentrations had to be used for the measurements).
.
diisopropyl-carbodiimide (or EDCI HCl+Et3N) and
10 mM of 1-hydroxybenzotriazole in anhydrous MeCN
as solvent. The reaction mixture was stirred at 4ꢀC for
3±9 h (TLC control). The solvent was evaporated in
vacuo and the residue taken up in ethyl acetate (50 mL),
poured into a 5% solution of sodium bicarbonate
(50 mL) and extracted with ethyl acetate. The combined
organic layers were dried over sodium sulfate and ®l-
tered, and the solvent removed in vacuo. In many cases
the compounds of type 4 precipitated, were ®ltered,
dried and deprotected at the a-amino- and N-1 imida-
zolic moieties in the following way. The crude 4 was
dissolved in 20 mL of CH2Cl2, treated with 4.8 mL of
tri¯uoroacetic acid (TFA) and stirred for 10 min at 0ꢀC.
The solvent was evaporated in vacuo and the syrup
obtained dissolved in 20 mL of dioxane and treated with
25 mL of a 4 M HCl solution in dioxane, followed by
heating at 40ꢀC for 6±8 h (TLC control). The solvent
was then evaporated under reduced pressure, the residue
taken up in 50 mL of a 5% solution of sodium bicarbo-
nate and the trityl sulfenyl chloride formed during the
deprotection step extracted in 2Â50 mL of Et2O. The
water phase was evaporated in vacuo to a small volume,
when generally crude compounds A1±A24 precipitated
by letting the mixture stand at 4ꢀC overnight. The pure
compounds A1±A24 were obtained by means of pre-
parative HPLC (C18 reversed-phase m-Bondapack or
Dynamax-60A (25Â250 mm) columns; 90% acetoni-
trile/8% methanol/2% water, 30 mL/min).
Preparation of N-1-tritylsulfenyl-histamine 3. Amounts
of 5.55 g (50 mM) of Hst and 23.15 g (50 mM) of tetra-
bromophthalic anhydride were suspended in 300 mL of
dry toluene and re¯uxed under Dean±Stark conditions
until water was separated (generally 2±3 h). The solvent
was evaporated in vacuo, the crude product dissolved in
150 mL of anhydrous acetonitrile and treated with
15.5 g (50 mM) of tritylsulfenyl chloride and 6.95 mL
(50 mM) of triethylamine. The mixture was stirred at
room temperature for 3 h (TLC control), then the sol-
vent was evaporated and the crude product 2 stirred
with 100 mL of water and ice. The tan precipitate
obtained was ®ltered, dried and used directly in the
deprotection step. Hydrazinolysis was e€ected by dis-
solving the above mentioned precipitate in 200 mL of
ethanol and addition of 15 mL of hydrazinium hydr-
oxide, followed by stirring for 5 h at room temperature.
Then the solvent was evaporated, a small excess of 2 N
HCl solution was added, the precipitated tetrabromo-
phthalhydrazide was ®ltered and discarded, whereas the
4-[ꢀ-(Glycylamido)-ethyl]-1H-imidazole A1. White
crystals, mp 279±281ꢀC (dec.); IR (KBr), cm 1: 1287
(amide III), 1579 (amide II), 1710 (amide I), 3060 (NH);
1H NMR (TFA), d, ppm: 2.49 (t, 2H, J=7.0, Hst CH2);
2.99 (t, 2H, J=7.0, Hst CONHCH2); 3.67 (s, 2H, CH2
of Gly); 7.34 (m, 1H, imidazole CH); 8.35 (s, 1H, imida-
zole CH); 13C NMR (TFA), d, ppm: 33.3 (s, CH2 of Hst);
37.9 (s, CH2 of Hst); 40.8 (s, CH2 of Gly); 122.4 (s, C-4
of Hst); 134.8 (s, C-5 of Hst); 137.3 (s, C-2 of Hst); 167.1
(CONH). Anal. found: C, 49.78; H, 7.20; N, 33.24%;
C7H12N4O requires: C, 49.99; H, 7.19; N, 33.31%.
4-[ꢀ-(ꢁ-Alanylamido)-ethyl]-1H-imidazole A2. White
crystals, mp 266±267ꢀC; IR (KBr), cm 1: 1285 (amide

2920
C. T. Supuran, A. Scozzafava / Bioorg. Med. Chem. 7 (1999) 2915±2923
1
III), 1574 (amide II), 1710 (amide I), 3060 (NH); H
3
J=7.0, Hst CH2); 3.01 (t, 2H, J=7.0, Hst CONHCH2);
3.75 (d, 3JHH=4.3, 1H, NHCHCH of Val), 7.34 (m, 1H,
imidazole CH); 8.35 (s, 1H, imidazole CH); 13C NMR
(TFA), d, ppm: 22.3 (s, CH(CH3)2 of Val), 33.3 (s, CH2
of Hst); 34.0 (s, CH(CH3)2 of Val), 37.9 (s, CH2 of Hst);
64.4 (s, NHCHCH of Val), 122.4 (s, C-4 of Hst); 134.7
(s, C-5 of Hst); 137.3 (s, C-2 of Hst); 167.9 (CONH).
Anal. found: C, 57.15; H, 8.94; N, 26.53%; C10H18N4O
requires: C, 57.12; H, 8.63; N, 26.64%.
NMR (TFA), d, ppm: 1.84 (d, JHH=6.5, 3H, CHCH3
of Ala), 2.49 (t, 2H, J=7.0, Hst CH2); 3.01 (t, 2H,
J=7.0, Hst CONHCH2); 3.98 (q, 1H, CH of Ala); 7.34
(m, 1H, imidazole CH); 8.35 (s, 1H, imidazole CH); 13C
NMR (TFA), d, ppm: 22.1 (s, CHCH3 of Ala); 33.3 (s,
CH2 of Hst); 37.9 (s, CH2 of Hst); 34.5 (s, CHCH3 of
Ala); 122.4 (s, C-4 of Hst); 134.8 (s, C-5 of Hst); 137.3
(s, C-2 of Hst); 167.9 (CONH). Anal. found: C, 52.71;
H, 7.42; N, 30.39%; C8H14N4O requires: C, 52.73; H,
7.74; N, 30.75%.
4-[ꢀ-(Leucylamido)-ethyl]-1H-imidazole A7. White crys-
tals, mp 251±253ꢀC; IR (KBr), cm 1: 1280 (amide III),
1
4-[ꢀ-(ꢀ-Alanylamido)-ethyl]-1H-imidazole A3. White
crystals, mp 278±279ꢀC (dec.); IR (KBr), cm 1: 1284
(amide III), 1575 (amide II), 1710 (amide I), 3060 (NH);
1H NMR (TFA), d, ppm: 2.49 (t, 2H, J=7.0, Hst CH2);
1575 (amide II), 1713 (amide I), 3060 (NH); H NMR
3
(TFA), d, ppm: 1.43 (d, JHH=6.7, 6H, CH(CH3)2 of
Leu), 1.55 (m, 2H, CH2 of Leu); 2.54 (t, 2H, J=7.0, Hst
CH2); 3.01 (t, 2H, J=7.0, Hst CONHCH2); 3.70 (m,
1H, NHCHCH2 of Leu), 4.23 (m, 1H, CHCH2-
CH(CH3)2 of Leu), 7.34 (m, 1H, imidazole CH); 8.35 (s,
1H, imidazole CH); 13C NMR (TFA), d, ppm: 21.7 (s,
CH(CH3)2 of Leu), 31.9 (s, CH2 of Leu); 33.3 (s, CH2 of
Hst); 34.0 (s, CH(CH3)2 of Leu), 37.9 (s, CH2 of Hst);
56.2 (s, NHCHCH2 of Leu), 122.4 (s, C-4 of Hst); 134.6
(s, C-5 of Hst); 137.3 (s, C-2 of Hst); 167.6 (CONH).
Anal. found: C, 58.98; H, 8.64; N, 24.70%; C11H20N4O
requires: C, 58.90; H, 8.99; N, 24.98%.
3
2.73 (t, JHH=6.6, 1H, CH2CH2CO of b-Ala); 2.99 (t,
3
2H, J=7.0, Hst CONHCH2); 3.27 (t, JHH=6.7, 1H,
3
CH2CH2CO of b-Ala), 3.43 (t, JHH=6.3, 2H, CH2-
CH2CO of b-Ala), 7.34 (m, 1H, imidazole CH); 8.35 (s,
1H, imidazole CH); 13C NMR (TFA), d, ppm: 33.3 (s,
CH2 of Hst); 37.5 (s, NHCH2CH2 of b-Ala), 37.9 (s,
CH2 of Hst); 40.9 (s, CH2CH2CO of b-Ala), 122.4 (s, C-
4 of Hst); 134.8 (s, C-5 of Hst); 137.3 (s, C-2 of Hst);
167.8 (CONH). Anal. found: C, 52.90; H, 7.68; N,
30.51%; C8H14N4O requires: C, 52.73; H, 7.74; N,
30.75%.
4-[ꢀ-(Isoleucylamido)-ethyl]-1H-imidazole A8. White
crystals, mp 221±223ꢀC; IR (KBr), cm 1: 1280 (amide
1
4-[ꢀ-(ꢂ-Aminobutyramido)-ethyl]-1H-imidazole A4.
:
III), 1575 (amide II), 1715 (amide I), 3060 (NH); H
3
White crystals, mp 256±257ꢀC (dec.); IR (KBr), cm
1287 (amide III), 1579 (amide II), 1710 (amide I), 3060
NMR (TFA), d, ppm: 1.15 (d, JHH=6.5, 3H, CH3 of
Ile), 1.21 (t, 3H, JHH=6.7, CH3 of Et moiety of Ile);
1
3
(NH); 1H NMR (TFA), d, ppm: 2.49 (t, 2H, J=7.0, Hst
CH2); 2.75 (t, JHH=6.5, 1H, (CH2)2CH2CO of
1.54 (m, 2H, CH2 of Ile); 2.54 (t, 2H, J=7.0, Hst CH2);
3.01 (t, 2H, J=7.0, Hst CONHCH2); 3.22 (m, 1H,
EtCH(Me) of Ile); 3.75 (m, 1H, NHCHCH of Ile),
7.36 (m, 1H, imidazole CH); 8.35 (s, 1H, imidazole CH);
13C NMR (TFA), d, ppm: 21.9 (s, CHCH3 of Ile), 22.5
(s, CH3CH2 of Ile); 31.4 (s, CH2 of Ile); 33.3 (s, CH2 of
Hst); 34.0 (s, CH(CH3)2 of Leu), 37.9 (s, CH2 of Hst);
46.4 (s, EtCH(Me) of Ile), 55.0 (s, NHCHCH2 of Ile),
122.4 (s, C-4 of Hst); 134.7 (s, C-5 of Hst); 137.3 (s, C-2
of Hst); 167.6 (CONH). Anal. found: C, 59.15; H, 8.75;
N, 24.83%; C11H20N4O requires: C, 58.90; H, 8.99; N,
24.98%.
3
GABA); 2.99 (t, 2H, J=7.0, Hst CONHCH2); 3.27 (t,
3JHH=6.7, 1H, (CH2)2CH2CO of GABA), 3.31±3.56
(m, 4H, (CH2)2CH2CO of GABA), 7.36 (m, 1H, imida-
zole CH); 8.35 (s, 1H, imidazole CH); 13C NMR (TFA),
d, ppm: 33.3 (s, CH2 of Hst); 37.3 (s, NHCH2CH2 of
GABA), 37.9 (s, CH2 of Hst); 40.9 (s, CH2CH2CH2CO
of GABA), 41.3 (s, CH2CH2CH2CO of GABA), 122.4
(s, C-4 of Hst); 134.8 (s, C-5 of Hst); 137.3 (s, C-2 of
Hst); 167.5 (CONH). Anal. found: C, 54.96; H, 8.24; N,
28.54%; C9H16N4O requires: C, 55.08; H, 8.22; N,
28.55%.
4-[ꢀ-(Asparaginylamido)-ethyl]-1H-imidazole A9. White
crystals, mp 254±255ꢀC; IR (KBr), cm 1: 1280 and 1295
(amide III), 1572 (amide II), 1710 and 1723 (amide I),
3060 (NH); 1H NMR (TFA), d, ppm: 2.54 (t, 2H,
J=7.0, Hst CH2); 2.59 (d, 2H, 3JHH=6.3, CH2 of Asn),
3.01 (t, 2H, J=7.0, Hst CONHCH2); 4.55 (m, 1H,
NHCHCH2 of Asn), 7.33 (m, 1H, imidazole CH); 8.35
(s, 1H, imidazole CH); 13C NMR (TFA), d, ppm: 30.1
(s, CHCH2 of Asn), 33.3 (s, CH2 of Hst); 37.9 (s, CH2 of
Hst); 70.4 (s, CHCH2 of Asn), 122.4 (s, C-4 of Hst);
134.7 (s, C-5 of Hst); 137.3 (s, C-2 of Hst); 177.0
(CONH); 178.9 (s, CONH2 of Asn). Anal. found: C,
48.12; H, 6.86; N, 31.05%; C9H15N5O2 requires: C,
47.99; H, 6.71; N, 31.09%.
4 - [[ꢀ - (Glycyl)glycylamido] - ethyl] - 1H - imidazole A5.
White crystals, mp 229±230ꢀC; IR (KBr), cm 1: 1290
(amide III), 1584 (amide II), 1715 (amide I), 3060 (NH);
1H NMR (TFA), d, ppm: 2.49 (m, 6H, J=7.0, Hst
CH2+2CH2 of GlyGly); 2.99 (t, 2H, J=7.0, Hst
CONHCH2); 7.34 (m, 1H, imidazole CH); 8.35 (s, 1H,
imidazole CH); 13C NMR (TFA), d, ppm: 33.3 (s, CH2
of Hst); 35.8 (s, CH2 of GlyGly); 37.9 (s, CH2 of Hst);
122.4 (s, C-4 of Hst); 134.7 (s, C-5 of Hst); 137.3 (s, C-2
of Hst); 167.1 (CONH); 172.5 (CONH). Anal. found: C,
48.10; H, 6.62; N, 30.97%; C9H15N5O2 requires: C,
47.99; H, 6.71; N, 31.09%.
4-[ꢀ-(Valylamido)-ethyl]-1H-imidazole A6. White crys-
tals, mp 256±258ꢀC; IR (KBr), cm 1: 1280 (amide III),
4-[ꢀ-(Glutaminylamido)-ethyl]-1H-imidazole A10. White
crystals, mp 259±260ꢀC; IR (KBr), cm 1: 1280 and 1293
(amide III), 1575 (amide II), 1710 and 1724 (amide I),
3060 (NH); 1H NMR (TFA), d, ppm: 2.54 (t, 2H,
1
1573 (amide II), 1710 (amide I), 3060 (NH); H NMR
3
(TFA), d, ppm: 1.11 (d, JHH=6.7, 6H, CH(CH3)2 of
Val), 2.29±2.50 (m, 1H, CH(CH3)2 of Val), 2.54 (t, 2H,

C. T. Supuran, A. Scozzafava / Bioorg. Med. Chem. 7 (1999) 2915±2923
2921
J=7.0, Hst CH2); 2.55±2.63 (m, 2H, CH2 of Gln), 3.01
(t, 2H, J=7.0, Hst CONHCH2); 3.05±3.34 (m, 2H, CH2
of Gln), 4.61 (m, 1H, NHCHCH2 of Gln), 7.34 (m, 1H,
imidazole CH); 8.35 (s, 1H, imidazole CH); 13C NMR
(TFA), d, ppm: 30.5 (s, CHCH2CH2 of Gln), 33.3 (s,
CH2 of Hst); 37.9 (s, CH2 of Hst); 44.7 (s, CHCH2CH2
of Gln), 72.6 (s, CHCH2CH2 of Gln), 122.4 (s, C-4 of
Hst); 134.8 (s, C-5 of Hst); 137.3 (s, C-2 of Hst); 177.2
(CONH); 179.5 (s, CH2CONH2 of Gln). Anal. found:
C, 49.98; H, 7.35; N, 29.18%; C10H17N5O2 requires: C,
50.20; H, 7.16; N, 29.27%.
4 - [ꢀ - (Phenylglycylamido) - ethyl] - 1H - imidazole A14.
White crystals, mp 256±257ꢀC (dec.); IR (KBr), cm
1
:
1284 (amide III), 1580 (amide II), 1710 (amide I), 3060
(NH); 1H NMR (TFA), d, ppm: 2.49 (t, 2H, J=7.0, Hst
CH2); 2.99 (t, 2H, J=7.0, Hst CONHCH2); 4.08 (m,
1H, PhCH of Phg), 7.29±7.58 (m, 6H, Harom of Phe+
imidazole CH of Hst); 8.35 (s, 1H, imidazole CH); 13C
NMR (TFA), d, ppm: 33.3 (s, CH2 of Hst); 37.9 (s, CH2
of Hst); 59.9 (s, PhCH of Phg), 122.4 (s, C-4 of Hst);
133.9 (s, Cmeta of Phg), 134.5 (s, Cortho of Phg), 134.8 (s,
C-5 of Hst); 137.3 (s, C-2 of Hst); 141.5 (s, Cipso of Phe),
166.9 (CONH). Anal. found: C, 64.15; H, 6.28; N, 22.70%;
C13H16N4O requires: C, 63.92; H, 6.60; N, 22.93%.
4-[ꢀ-(Arginylamido)-ethyl]-1H-imidazole A11. White
crystals, mp 277±279ꢀC (dec.); IR (KBr), cm 1: 1285
(amide III), 1584 (amide II), 1712 (amide I), 3060 (NH);
1H NMR (TFA), d, ppm: 1.70±2.00 (m, 2H, CHCH2-
CH2 of Arg), 2.49 (t, 2H, J=7.0 Hz, Hst CH2); 2.50±
4-[ꢀ-(Phenylalanylamido)-ethyl]-1H-imidazole A15.
White crystals, mp 239±241ꢀC (dec.); IR (KBr), cm
1
:
1280 (amide III), 1581 (amide II), 1710 (amide I), 3060
(NH); 1H NMR (TFA), d, ppm: 2.49 (t, 2H, J=7.0, Hst
CH2); 2.99 (t, 2H, J=7.0, Hst CONHCH2); 3.10±3.55
3
2.58 (m, 2H, CHCH2CH2 of Arg), 2.75 (t, JHH=6.5,
1H, (CH2)2CH2CO of Arg); 2.99 (t, 2H, J=7.0 Hz, Hst
CONHCH2); 3.30±3.45 (m, 2H, CH2CH2NH of Arg),
3.45±3.60 (m, 1H, CH2CH(NH)CO of Arg), 7.36 (m,
1H, imidazole CH); 8.35 (s, 1H, imidazole CH); 13C
NMR (TFA), d, ppm: 29.7 (s, CH2CH2CH2 of Arg),
33.3 (s, CH2 of Hst); 35.4 (s, CHCH2CH2 of Arg), 37.9
(s, CH2 of Hst); 45.7 (s, CH2CH2NH of Arg), 59.8 (s,
CH2CH(NH)CO2H of Arg), 122.4 (s, C-4 of Hst); 134.6
(s, C-5 of Hst); 137.3 (s, C-2 of Hst); 161.5 (s,
NHC(ˆNH)NH2 of Arg), 170.8 (CONH). Anal. found:
C, 49.19; H, 7.81; N, 36.69%; C11H21N7O requires: C,
49.42; H, 7.92; N, 36.68%.
3
(m, 2H, CH2CH of Phe), 4.08 (dd, JHH=5.0,
3JHH=7.8, 1H, CH2CH of Phe), 7.29±7.58 (m, 6H,
Harom of Phe+imidazole CH of Hst); 8.35 (s, 1H, imi-
dazole CH); 13C NMR (TFA), d, ppm: 33.3 (s, CH2 of
Hst); 37.9 (s, CH2 of Hst); 41.7 (s, CH2CH of Phe), 59.3
(s, CH2CH of Phe), 122.4 (s, C-4 of Hst); 133.8 (s, Cmeta
of Phe), 134.4 (s, Cortho of Phe), 134.8 (s, C-5 of Hst);
137.3 (s, C-2 of Hst); 141.5 (s, Cipso of Phe), 166.9
(CONH). Anal. found: C, 65.21; H, 7.13; N, 21.54%;
C14H18N4O requires: C, 65.09; H, 7.02; N, 21.69%.
4-[ꢀ-(Tryptophanylamido)-ethyl]-1H-imidazole A16.
White crystals, mp 208±210ꢀC; IR (KBr), cm 1: 1280
(amide III), 1581 (amide II), 1710 (amide I), 3060 (NH);
4-[ꢀ-[ꢁ-(Lysylamido)]-ethyl]-1H-imidazole A12. White
crystals, mp 223±224ꢀC; IR (KBr), cm 1: 1281 (amide
1
III), 1579 (amide II), 1711 (amide I), 3060 (NH); H
NMR (TFA), d, ppm: 1.66±2.20 (m, 6H, CH(CH2)3CH2
of Lys), 2.49 (t, 2H, J=7.0 Hz, Hst CH2); 2.99 (t, 2H,
1H NMR (TFA), d, ppm: 2.49 (t, 2H, J=7.0, Hst CH2);
3
2.99 (t, 2H, J=7.0, Hst CONHCH2); 3.44 (dd, JHH
2
=
9.0, JHH=14.6, 1H, CH2CH of Trp), 3.65 (dd,
3
2
J=7.0 Hz, Hst CONHCH2); 3.13 (t, JHH=6.7, 2H,
3
3JHH=4.1, JHH=15.0, 1H, CH2CH of Trp), 4.14 (dd,
3
CH2CH2NH2 of Lys), 3.84 (t, JHH=6.7, 1H, CH2CH-
3JHH=4.3, JHH=8.0, 1H, CH2CH of Trp), 7.22±7.82
(NH)CO of Lys), 7.36 (m, 1H, imidazole CH); 8.35 (s,
1H, imidazole CH); 13C NMR (TFA), d, ppm: 26.6 (s,
H2NCH2(CH2)3 of Lys), 31.4 (s, H2NCH2(CH2)3 of
Lys), 33.3 (s, CH2 of Hst); 34.8 (s, H2NCH2(CH2)3 of
Lys), 37.9 (s, CH2 of Hst); 43.8 (s, H2NCH2(CH2)3 of
Lys), 58.8 (s, CH2CH(NH)CO of Lys), 122.4 (s, C-4 of
Hst); 134.8 (s, C-5 of Hst); 137.3 (s, C-2 of Hst); 171.2
(CONH). Anal. found: C, 55.17; H, 8.70; N, 29.13%;
C11H21N5O requires: C, 55.21; H, 8.84; N, 29.26%.
(m, 6H, Harom of Trp+1H, imidazole CH of Hst), 8.35
(s, 1H, imidazole CH); 13C NMR (TFA), d, ppm: 31.6
(s, CH2CH of Trp), 33.3 (s, CH2 of Hst); 37.9 (s, CH2 of
Hst); 58.8 (s, CH2CH of Trp), 113.9 (s, C-2 of Trp),
116.9 (s, C-7 of Trp), 122.4 (s, C-4 of Hst); 123.4 (s, C-5
of Trp), 124.3 (s, C-6 of Trp), 126.8 (s, C-4 of Trp),
129.0 (s, C-1 of Trp), 132.1 (s, C-8 of Trp), 134.7 (s, C-5
of Hst); 137.3 (s, C-2 of Hst); 141.2 (s, C-3 of Trp),
168.2 (CONH). Anal. found: C, 64.56; H, 6.41; N,
23.47%; C16H19N5O requires: C, 64.63; H, 6.44; N,
23.55%.
4-[ꢀ-(Histidylamido)-ethyl]-1H-imidazole A13. White
crystals, mp 270±272ꢀC (dec.); IR (KBr), cm 1: 1282
(amide III), 1585 (amide II), 1710 (amide I), 3060 (NH);
1H NMR (TFA), d, ppm: 2.49 (t, 2H, J=7.0, Hst CH2);
2.99 (t, 2H, J=7.0, Hst CONHCH2); 3.32±3.46 (m, 2H,
CHCH2 of His), 4.01±4.08 (m, 1H, CHCH2 of His), 7.34
(m, 1H, imidazole CH of Hst); 7.46 (s, 1H, CH-5 of
His), 8.35 (s, 1H, imidazole CH); 8.51 (s, 1H, CH-2 of
His); 13C NMR (TFA), d, ppm: 33.3 (s, CH2 of Hst);
37.9 (s, CH2 of Hst); 40.9 (s, CH2 of His); 58.6 (s,
CH2CH of His), 122.2 (s, C-4 of His); 122.4 (s, C-4 of
Hst); 134.1 (s, C-5 of His); 134.6 (s, C-5 of Hst); 137.2
(s, C-2 of Hst); 137.5 (s, C-2 of His); 167.1 (CONH).
Anal. found: C, 53.21; H, 6.18; N, 33.79%; C11H16N6O
requires: C, 53.21; H, 6.50; N, 33.85%.
4-[ꢀ-(Prolylamido)-ethyl]-1H-imidazole A17. Mp 276±
278ꢀC (dec.); IR (KBr), cm 1: 1286 (amide III), 1589
(amide II), 1718 (amide I), 3060 (NH); 1H NMR (TFA),
d, ppm: 1.18±1.38 (m, 1H, HCH of Pro); 1.55±1.65 (m,
1H, HCH); 1.70±1.85 (m, 2H, CH2 of Pro); 2.49 (t, 2H,
J=7.0, Hst CH2); 2.99 (t, 2H, J=7.0, Hst CONHCH2);
3.16±3.30 (m, 2H, CH2N of Pro); 3.75±3.80 (m, 1H,
CHCO of Pro), 7.34 (m, 1H, H-5 of Hst); 8.35 (s, 1H,
H-2 of Hst); 13C NMR (TFA), d, ppm: 15.6 (s, CH2 of
Pro); 21.3 (s, CH2 of Pro); 33.3 (s, CH2 of Hst); 37.9 (s,
CH2 of Hst); 46.9 (s, CH2N of Pro); 64.5 (s, CHCO of
Pro), 122.4 (s, C-4 of Hst); 134.8 (s, C-5 of Hst); 137.3
(s, C-2 of Hst); 170.3 (s, Pro-CONH). Anal. found: C,

2922
C. T. Supuran, A. Scozzafava / Bioorg. Med. Chem. 7 (1999) 2915±2923
57.60; H, 7.61; N, 26.82% C10H16N4O requires: C,
57.67; H, 7.74; N, 26.90%.
4-[ꢀ-(ꢀ-Alanyl-histidylamido)-ethyl]-1H-imidazole A22.
Mp 240±243ꢀC; IR (KBr), cm 1: 1288 (amide III),
1592 (amide II), 1715 (amide I), 3060 (NH); H NMR
1
4-[ꢀ-(Pipecolylamido)-ethyl]-1H-imidazole A18. Mp
237±238ꢀC; IR (KBr), cm 1: 1284 (amide III), 1589
(amide II), 1712 (amide I), 3060 (NH); 1H NMR (TFA),
d, ppm: 1.19±1.43 (m, 1H, HCH of Pip); 1.50±1.65 (m,
1H, HCH); 1.69±1.87 (m, 4H, 2CH2 of Pip); 2.49 (t, 2H,
J=7.0, Hst CH2); 2.99 (t, 2H, J=7.0, Hst CONHCH2);
3.16±3.34 (m, 2H, CH2N of Pip); 3.70±3.83 (m, 1H,
CHCO of Pip), 7.33 (m, 1H, H-5 of Hst); 8.35 (s, 1H, H-
2 of Hst); 13C NMR (TFA), d, ppm: 15.6 (s, CH2 of
Pip); 18.0 (s, CH2 of Pip); 21.3 (s, CH2 of Pip); 33.3 (s,
CH2 of Hst); 37.9 (s, CH2 of Hst); 46.4 (s, CH2N of
Pip); 64.9 (s, CHCO of Pip), 122.4 (s, C-4 of Hst); 134.8
(s, C-5 of Hst); 137.3 (s, C-2 of Hst); 170.0 (s, Pip-
CONH). Anal. found: C, 59.63; H, 7.89; N, 25.16%;
C11H18N4O requires: C, 59.44; H, 8.16; N, 25.20%.
(TFA), d, ppm: 2.49 (t, 2H, J=7.0, Hst CH2); 2.99 (t,
2H, J=7.0, Hst CONHCH2); 2.79±2.88 (m, 2H, CH2 of
b-Ala), 3.11±3.26 (m, 2H, CH2 of b-Ala), 3.34±3.45 (m,
2H, CHCH2 of His), 4.57±4.63 (m, 1H, CHCH2 of His),
7.33 (s, 2H, CH-5 of His+Hst), 8.35 (s, 2H, CH-2 of
His+Hst); 13C NMR (TFA), d, ppm: 33.3 (s, CH2 of
His), 33.4 (s, CH2 of Hst); 37.4 (s, NHCH2CH2 of b-
Ala), 37.9 (s, CH2 of Hst); 40.8 (s, CH2CH2CO of b-
Ala), 59.6 (s, CHCH2 of His), 122.2 (s, C-4 of His),
122.5 (s, C-4 of Hst); 134.2 (s, C-5 of His), 134.8 (s, C-5
of Hst); 137.2 (s, C-2 of His), 137.5 (s, C-2 of Hst); 175.6
(s, CH2CO of b-Ala), 176.7 (s, CONH of His). Anal.
found: C, 52.44; H, 6.70; N, 30.53%; C14H21N7O2
requires: C, 52.65; H, 6.63; N, 30.70%.
4-[ꢀ-(Phenylalanyl-prolylamido)-ethyl]-1H-imidazole A23.
Mp 233±234ꢀC (dec.); IR (KBr), cm 1: 1289 (amide III),
1590 (amide II), 1717 (amide I), 3060 (NH); H NMR
4-[ꢀ-(Nipecotylamido)-ethyl]-1H-imidazole A19. Mp
244±245ꢀC; IR (KBr), cm 1: 1280 (amide III), 1593
(amide II), 1710 (amide I), 3060 (NH); 1H NMR (TFA),
d, ppm: 1.24±1.67 (m, 4H, CH2CH2 of Nip); 2.49 (t, 2H,
J=7.0, Hst CH2); 2.99 (t, 2H, J=7.0, Hst CONHCH2);
3.16±3.34 (m, 4H, CH2NCH2 of Nip); 3.72±3.88 (m, 1H,
CHCO of Nip), 7.33 (m, 1H, H-5 of Hst); 8.35 (s, 1H,
H-2 of Hst); 13C NMR (TFA), d, ppm: 16.3 (s, CH2 of
Nip); 21.0 (s, CH2 of Nip); 33.3 (s, CH2 of Hst); 37.9 (s,
CH2 of Hst); 46.4 (s, CH2N of Nip); 47.5 (s, NCH2 of
Nip); 53.6 (s, CHCO of Nip), 122.4 (s, C-4 of Hst); 34.8
(s, C-5 of Hst); 135.1 (s, Cortho of CH3C6H4), 137.3 (s,
C-2 of Hst); 170.0 (s, Nip-CONH). Anal. found: C,
59.40; H, 8.05; N, 25.11%; C11H18N4O requires: C,
59.44; H, 8.16; N, 25.20%.
1
(TFA), d, ppm: 1.19±1.40 (m, 1H, HCH of Pro); 1.55±
1.69 (m, 1H, HCH); 1.75±1.85 (m, 2H, CH2 of Pro);
2.49 (t, 2H, J=7.0, Hst CH2); 2.99 (t, 2H, J=7.0, Hst
CONHCH2); 3.10±3.56 (m, 2H, CH2CH of Phe), 3.20±
3.35 (m, 2H, CH2N of Pro); 3.75±3.80 (m, 1H, CHCO
3
3
of Pro), 4.12 (dd, JHH=5.0, JHH=7.8, 1H, CH2CH
of Phe), 7.29±7.58 (m, 6H, Harom of Phe+H-5 of Hst);
8.34 (s, 1H, CH-2 of Hst); 13C NMR (TFA), d, ppm:
15.5 (s, CH2 of Pro); 21.5 (s, CH2 of Pro); 33.5 (s,
CH2 of Hst); 37.9 (s, CH2 of Hst); 41.6 (s, CH2CH of
Phe), 46.9 (s, CH2N of Pro); 59.5 (s, CHCH2 of Phe),
64.5 (s, CHCO of Pro), 122.4 (s, C-4 of Hst); 130.2 (s,
Cpara of Phe), 133.9 (s, Cmeta of Phe), 134.7 (s, Cortho
of Phe); 134.6 (s, C-5 of Hst); 137.3 (s, C-2 of Hst);
141.5 (s, Cipso of Phe), 172.3 (s, Phe-CONH); 174.0
(Pro-CONH). Anal. found: C, 64.35; H, 7.15; N,
19.58%; C19H25N5O2 requires: C, 64.20; H, 7.09; N,
19.70%.
4-[ꢀ-(Isonipecotylamido)-ethyl]-1H-imidazole A20. Mp
270±271ꢀC (dec.); IR (KBr), cm 1: 1277 (amide III),
1
1591 (amide II), 1711 (amide I), 3060 (NH); H NMR
(TFA), d, ppm: 1.90±2.33 (m, 8H, 2 CH2CH2 of Inp);
2.49 (t, 2H, J=7.0, Hst CH2); 2.99 (t, 2H, J=7.0, Hst
CONHCH2); 3.13±3.39 (m, 1H, CHCO of Inp), 7.33 (m,
1H, H-5 of Hst); 8.35 (s, 1H, H-2 of Hst); 13C NMR
(TFA), d, ppm: 21.6 (s, CH2 of Inp); 33.3 (s, CH2 of
Hst); 37.9 (s, CH2 of Hst); 47.0 (s, NCH2 of Inp); 53.2
(s, CHCO of Inp), 122.4 (s, C-4 of Hst); 134.8 (s, C-5 of
Hst); 137.3 (s, C-2 of Hst); 170.6 (s, Inp-CONH). Anal.
found: C, 59.28; H, 8.15; N, 25.00%; C11H18N4O
requires: C, 59.44; H, 8.16; N, 25.20%.
4-[ꢀ-(Prolyl-glycylamido)-ethyl]-1H-imidazole A24. Mp
235±237ꢀC; IR (KBr), cm 1: 1289 (amide III), 1590
(amide II), 1717 (amide I), 3060 (NH); 1H NMR (TFA),
d, ppm: 1.18±1.38 (m, 1H, HCH of Pro); 1.55±1.65 (m,
1H, HCH); 1.70±1.85 (m, 2H, CH2 of Pro); 2.49 (t, 2H,
J=7.0, Hst CH2); 2.99 (t, 2H, J=7.0, Hst CONHCH2);
3.16±3.30 (m, 2H, CH2N of Pro); 3.67 (s, 2H, CH2 of
Gly), 3.75±3.80 (m, 1H, CHCO of Pro), 7.34 (m, 1H, H-
5 of Hst); 8.35 (s, 1H, H-2 of Hst); 13C NMR (TFA), d,
ppm: 15.6 (s, CH2 of Pro); 21.3 (s, CH2 of Pro); 33.5 (s,
CH2 of Hst); 37.9 (s, CH2 of Hst); 40.8 (s, CH2 of Gly),
46.4 (s, CH2N of Pro); 64.7 (s, CHCO of Pro), 122.4 (s,
C-4 of Hst); 134.7 (s, C-5 of Hst); 137.3 (s, C-2 of Hst);
175.6 (s, Pro-CONH); 176.3 (s, CONH of Gly). Anal.
found: C, 54.28; H, 7.27; N, 26.09%; C12H19N5O2
requires: C, 54.33; H, 7.22; N, 26.40%.
4-[ꢀ-(Glycyl-histidylamido)-ethyl]-1H-imidazole A21. Mp
220±221ꢀC; IR (KBr), cm 1: 1288 (amide III), 1592
(amide II), 1715 (amide I), 3060 (NH); 1H NMR (TFA), d,
ppm: 2.49 (t, 2H, J=7.0, Hst CH2); 2.99 (t, 2H, J=7.0,
Hst CONHCH2); 3.67 (s, 2H, CH2 of Gly), 3.35±3.46 (m,
2H, CHCH2 of His), 4.59±4.67 (m, 1H, CHCH2 of His),
7.34 (s, 2H, CH-5 of His + Hst), 8.35 (s, 2H, CH-2 of His
+ Hst); 13C NMR (TFA), d, ppm: 33.3 (s, CH2 of Hst);
37.9 (s, CH2 of Hst); 40.5 (s, CH2 of Gly), 59.6 (s, CHCH2
of His), 122.2 (s, C-4 of His), 122.4 (s, C-4 of Hst); 132.0 (s,
C-5 of His), 134.8 (s, C-5 of Hst); 137.2 (s, C-2 of His),
137.4 (s, C-2 of Hst); 175.0 (s, CH2CO of Gly), 176.4 (s,
CONH of His). Anal. found: C, 51.21; H, 6.32; N, 32.10%;
C13H19N7O2 requires: C, 51.14; H, 6.27; N, 32.11%.
Acknowledgements
This research was ®nanced by the EU grant ERB
CIPDCT 940051. Thanks are addressed to Drs. M. A.
Ilies and B. Iorga for expert technical assistance.

C. T. Supuran, A. Scozzafava / Bioorg. Med. Chem. 7 (1999) 2915±2923
2923
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