Carbonic anhydrase activators - Part 21. Novel activators of isozymes I, II and IV incorporating carboxamido and ureido histamine moieties

Article abstract of DOI:10.1016/S0223-5234(00)00102-1

Reaction of histamine 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. Carboxamido derivatives were obtained by reaction of the key intermediate with carboxylic acid anhydrides, acyl chlorides or carboxylic acids in the presence of carbodiimides. Reaction of the same key intermediate with isocyanates, isothiocyanates, cyanamide or dicyandiamide afforded another series of compounds. Deprotection of the above-mentioned intermediates with hydrochloric acid in dioxane afforded two series of compounds, histamine derivatives possessing carboxamido, ureido, thioureido or guanidino moieties in their molecule. 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, h = human, b = bovine isozyme). 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 10-25 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. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00102-1

Eur. J. Med. Chem. 35 (2000) 31−39
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001021/FLA
31
Original article
Carbonic anhydrase activators – Part 21#. Novel activators of isozymes I, II
and IV incorporating carboxamido and ureido histamine moieties
Andrea Scozzafava, Claudiu T. Supuran*
Università degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Florence, Italy
Received 29 March 1999; revised 16 June 1999; accepted 17 June 1999
Abstract – Reaction of histamine 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.
Carboxamido derivatives were obtained by reaction of the key intermediate with carboxylic acid anhydrides, acyl chlorides or carboxylic acids
in the presence of carbodiimides. Reaction of the same key intermediate with isocyanates, isothiocyanates, cyanamide or dicyandiamide
afforded another series of compounds. Deprotection of the above-mentioned intermediates with hydrochloric acid in dioxane afforded two
series of compounds, histamine derivatives possessing carboxamido, ureido, thioureido or guanidino moieties in their molecule. 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, h = human, b = bovine isozyme). 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 10–25 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. © 2000 Éditions scientifiques et médicales Elsevier SAS
carbonic anhydrase / histamine / carboxylic acid derivatives / isocyanates / isothiocyanates / enzyme activators
1. Introduction
CA deficiency syndrome, a genetic disease of bone, brain
and kidney affecting a large enough number of pa-
tients [14]. 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
specific treatment for this condition is available up to
now. CA activators are also important for understanding
the CA catalytic and inhibition mechanisms [7–13].
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], epilepsy [3], gastric and
duodenal ulcers [4] or acid–base disequilibria [5] among
others. In contrast to inhibitors, activators of this enzyme
(for which at least eight different isozymes were isolated
up to now in higher vertebrates) [6] were much less
investigated. Only recently the X-ray crystallographic
structures of the first adducts of the physiologically
relevant isozyme II (hCA II) with the activators hista-
mine [7] and phenylalanine (in this case a tertiary com-
plex, in which azide is also bound to the Zn(II) ion) [8]
have been reported by this group. Furthermore, few other
QSAR [9–11] or synthetic chemistry [10–13] studies
were reported in the field of CA activators, although some
of these compounds might be used in the treatment of the
The lead molecule considered by us for obtaining
tighter binding CA activators was histamine 1 itself. As
seen from the X-ray co-ordinates 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 nitro-
gen 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 Nδ1 and Ne2
#See Ref. [1]
*Correspondence and reprints: cts@bio.polosci.unifi.it

32
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 centre of the active site, whereas histamine (numbered as His 264) is situated at the entrance to it, between residues His
64 and Gln 92. The figure was generated from the X-ray coordinates of the hCA II–histamine adduct reported by this group [7] with
the program RasMol for Windows 2.6. The coordinates of this structure are depositated in the Brookhaven Protein Database (PDB
entry 4TST).
atoms of the histidine imidazole ring are engaged in
hydrogen bonds with the side-chains of Asn 62, His 64,
Gln 92 and with Wat 152 [7]. Thus, it appeared of interest
to derivatize the lead at its aliphatic NH2 moiety, just in
order to exploit the energy of binding of such modified
groups with amino acid residues at the edge of the active
site. This approach has been successfully used both by
Whiteside’s [15] and our group [7, 16, 17] for the design
of tight-binding, isozyme-specific sulfonamide CA in-
hibitors. Moreover, recently we have reported some
sulfonylated derivatives of histamine (at the aliphatic
amino group) possessing high affinities for the three CA
isozymes mentioned above [1]. Thus, it appeared of
interest to explore other types of moieties that might be
attached at the aliphatic end of the molecule, and car-
boxamido, ureido and thioureido derivatives appeared of
interest due to the possible interactions of these highly
polarized groups with amino acid residues at the edge of
the active site, which presumably would lead to increased
stabilities of the enzyme–activator adducts.
In this paper we report the synthesis of a series of
carboxamido, ureido, thioureido and guanidino histamine
derivatives obtained by reaction of appropriately pro-
tected [1, 18] histamine with carboxylic acids, acyl chlo-
rides, carboxylic anhydrides, isocyanates, isothiocyanates
or cyanamide. 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 these series of
derivatives are also discussed.

33
Figure 2. Synthesis of CA activators A1–A39.
2. Chemistry
groups were used by us, making it possible to work in
much milder reaction conditions.
Reaction of histamine 1 with tetrabromophthalic anhy-
dride afforded the corresponding phthalimide, which was
further protected at the imidazole NH moiety with triphe-
nylmethanesulfenyl chloride (tritylsulfenyl chloride),
leading to 2. The key intermediate (N-1-tritylsulfenyl
histamine, 3) was easily obtained by room temperature
hydrazinolysis of 2, whereas its treatment with carboxylic
acid anhydrides, acyl halides in the presence of triethyl-
amine or carboxylic acids in the presence of carbodiimide
derivatives, afforded derivatives of type 4 in high yields.
These last compounds were then deprotected with 4 M
HCl-dioxane, giving the target compounds A1–A39.
Alternatively, another series of compounds was obtained
by treating N-1-tritylsulfenyl histamine 3 with aryl/alkyl
isocyanates/isothiocyanates, cyanamide or dicyandia-
mide, followed by deprotection of the imidazolic moiety
in a similar manner as that described above, leading to
derivatives of type B40–B49 (figure 2). The first proce-
dure mentioned above is relatively similar to that reported
recently by Wolin et al. [18] for the syntheses of some
sulfonylated homologues of histamine possessing H3
antagonistic properties, except that different protecting
Another two compounds, C1 and C2, were obtained by
the reaction of EDTA dianhydride or DTPA dianhydride
with histamine in molar ratios of 1:2 (figure 3).
3. Pharmacology
The new activators reported here were assayed against
three CA isozymes (table I) due to the fact that hCA I,
hCA II and bCA IV are the most abundant among the
eight CA’s isolated so far in higher vertebrates [6] on one
hand, and on the other one, the high activity forms hCA
II and bCA IV are considered to play the key physiologi-
cal function in many tissues in which CA activity is
present [2, 3, 6]. Although hCA I is the second most
abundant protein in blood (after serum albumin), its
physiological role is unknown [2, 4, 6]. Its activation
process has never been considered from the physiological
point of view up to now.
Thus, it was recently proven by this group that a
concentration as low as 10–7 M of histamine produces a
150% activation of hCA I [7]. Although hCA II, one of

34
alanine with hCA II, has this topic received again its
deserved importance, and some other types of high
affinity activators investigated mainly by this [7–13] and
Chegwidden’s [22] group.
The key intermediate for obtaining novel types of
activators reported in this paper, N-1-tritylsulfenyl hista-
mine 3, was obtained by well-known procedures involv-
ing the initial protection of the primary amine moiety by
means of phthalimide derivatives, followed by protection
of the imidazolic NH moiety with tritylsulfenyl chloride,
and hydrazinolysis of the phthalimido moiety in mild
conditions. The overall yield of the three steps was good
(around 80%) and the purification procedures quite
simple. Subsequent reaction of the key intermediate 3
with carboxylic acids or their derivatives [23, 24], or with
isocyanates/isothiocyanates/cyanamides [25], afforded
two series of N-tritylsulfenylated compounds, 4 and 5,
which were deprotected in standard conditions, leading
thus to the desired derivatives A1–A39 and B40–B49.
Alternatively, reaction of EDTA dianhydride or DTPA
dianhydride with histamine at molar ratios of 1:2 afforded
the bis-functionalized derivatives C1 and C2. All the new
compounds reported here have been characterized by IR,
1H- and 13C-NMR spectroscopy, as well as elemental
analysis (± 0.4% of the theoretical data, calculated for the
proposed formulae). Spectral data for one representative
example of each series are shown in the Experimental
protocols.
Figure 3. Structures of the potent CA activators C1 and C2.
the most powerful catalysts ever evolved in nature [4], is
not so abundant as hCA I [4, 6], this high activity isozyme
is present in virtually every cell [2, 4, 6] where it
catalyses the hydration of CO2 generated in metabolic
processes. In many tissues (such as ciliary processes
within the eye, gastric or pancreatic mucosa, kidneys,
etc.) it also participates in the processes of electrolyte
–
secretion, generating H+ or HCO3 ions (the products of
CO2 hydration) [2, 4]. Isozyme IV is the only membrane-
bound one, being very abundant in the lung, kidney,
gastro-intestinal tract and hepatocytes of vertebrates
among others [2, 4, 14]. Like hCA II, it is a high activity
form [4]. It is thus of considerable interest to search
activators (and obviously, also inhibitors) that would
react predominantly with one or the other of these
physiologically important enzymes. This should consti-
tute an interesting means for assessing the physiological
function of many of the presently known CA isozymes, as
well as for the development of agents able to activate CA
isozymes in patients affected by CA deficiency syn-
dromes [14].
The data of table I show significant differences be-
tween the investigated isozymes in their behaviour to-
wards both ‘classical’ activators, such as histamine 1, as
well as the new class of activators synthesized in the
present work. Thus, histamine 1 is a potent hCA I
activator and a relatively weak hCA II activator, whereas
isozyme bCA IV possesses an intermediate behaviour.
The most interesting finding of the present study is
represented by the high susceptibility of the cytosolic
isozyme, hCA II, to be activated by some of the deriva-
tives of histamines of types A–C, as compared to the lead
molecule (compounds with activation constants in the
0.10–0.15 µM range were frequently obtained). More-
over, the highly abundant and most prone to activation
(by histamine) isozyme hCA I, was also susceptible to
activation by the new derivatives reported here (with
constants in the nanomolar range for the most active
derivatives), but differences of activity are not so pro-
nounced as compared to the situation for the rapid
isozyme hCA II. bCA IV, on the other hand, had an
intermediate behaviour towards the new class of activa-
tors, with activation constants in the 0.01–0.10 µM range
for the most active of such compounds.
4. Results and discussion
Silverman’s laboratory [19] reported activation of hCA
II by histidine, and then considered 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 buffer [20]. We
explained in a previous publication the problems caused
by the above mentioned experimental protocols [21]; and
only recently, with the report of the first X-ray crystallo-
graphic data [7, 8] of adducts of histamine and phenyl-

35
Table I. CA isozymes I, II and IV activation with histamine 1 and its derivatives of types A1–A39, B40–B49 and C1, C2, together with their
synthetic methods.
No.
X
R
KA* (µM)
Synth. method
hCA Ia
hCA IIa
bCA IVb
1
A1
A2
A3
A4
A5
A6
A7
– histamine
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
O
O
O
O
O
S
S
S
NH
NH
–
–
CF3
CCl3
CHCl2
2
125
1.0
1.3
4.9
5.5
70
84
86
62
39
12
13
13
9
10
8
7
1.3
0.8
0.5
0.10
10
9
41
–
0.016
0.020
0.18
0.67
0.21
0.18
0.20
0.21
0.12
0.11
0.10
0.10
0.07
0.08
0.05
0.06
0.03
0.06
0.05
0.02
0.14
0.12
0.13
0.009
0.008
0.006
0.005
0.004
0.005
0.05
0.26
0.27
0.08
0.09
2.5
3.6
5.5
6.3
21
19
16
A
B
B
C
B
A
C
A
A
B
B
B
B
C
C
C
C
C
C
B
C
C
B
C
B
B
C
C
C
A
A
A
C
C
B
C
C
C
D
D
D
D
D
D
E
E
E
F
CH2Cl
CH3(CH2)10
HOOCCH2CH2
HOOC(CH2)4
cis-HOOCCH=CH
HOOCCH2OCH2
4-F-C6H4
2-Cl-C6H4
3-Cl-C6H4
2,4-Cl2C6H3
4-I-C6H4
A8
A9
14
13
A10
A11
A12
A13
A14
A15
A16
A17c
A18c
A19c
A20
A21
A22
A23
A24
A25
A26
A27d
A28e
A29f
A30
A31
A32
A33c
A34c
A35
A36
A37
A38
A39
B40
B41
B42
B43
B44
B45
B46
B47
B48
B49
C1
2.9
2.7
2.5
3.0
3.2
4.0
4.4
0.15
0.10
0.65
0.02
18
3-I-C6H4
2-I-C6H4
4-tsHN-C6H4
3-tsHN-C6H4
2-tsHN-C6H4
C6F5
1-naphthyl
2-naphthyl
2-thienyl
2-pyridyl
3-pyridyl
4-pyridyl
NTA
15
10
8
0.15
0.20
0.18
3.1
0.7
0.5
14
25
29
9
1.4
1.5
1.0
0.9
0.9
0.8
5.1
6.6
7.5
3.2
3.1
5.8
EDTA
DTPA
2-HOOC-C6Br4
2-HOOC-C6H4
2-HOOC-C6Hr11
ts-Nipecotinoyl
ts-Isonipecotinoyl
Ph2N
4-Cl-3-H2NO2S-C6H3
4-n-Pr2NSO2-C6H4
4-H2NO2S-C6H4
H2N
4-Cl-C6H4
3-Cl-C6H4
2,4-F2C6H3
2,4-Cl2C6H3
1-naphthyl
CH2=CHCH2
C6H5CONH
H
5
13
0.15
strong CA inhibitorg
0.04
1.9
4.2
strong CA inhibitorh
0.21
45
24
0.005
0.006
0.005
0.006
0.015
0.020
0.004
0.036
0.015
0.010
0.006
0.003
0.19
0.15
0.08
0.12
0.40
3.5
0.12
16
1.50
0.25
0.12
0.08
0.05
0.03
0.02
0.07
0.10
0.25
0.03
12
0.3
0.5
0.03
0.02
H
H2NC(=NH)-
–
–
G
–
–
C2
–
* Mean from at least two determinations by the esterase method [22]. Standard error was in the range of 5–10%; a Human cloned isozyme; b Purified from
bovine lung microsomes [21]; c ts = 4-Me-C6H4SO2NHCO-; d NTA = nitrilotriacetyl, (HOOC)2N-CO-; e EDTA = ethylenediaminotetraacetyl, (HOOCCH2)2N-
CH2CH2N(CH2COOH)CH2CO-; f DTPA = diethylenetriaminopetaacetyl, (HOOCCH2)2N-CH2CH2N (CH2COOH)CH2CH2N(CH2COOH)CH2CO-; g KI of: 32
nM (hCA I); 3.4 nM (hCA II) and 12 nM (bCA IV), respectively; h KI of: 35 nM (hCA I); 4.1 nM (hCA II) and 13 nM (bCA IV), respectively.
Synthetic methods: A: N-1-tritylsulfenyl-histamine (3) + (RCO)2O, followed by deprotection; B: N-1-tritylsulfenyl-histamine (3) + RCOCl/Et3N, followed by
deprotection; C: N-1-tritylsulfenyl-histamine (3) + RCOOH/carbodiimide/1-HO-benzotriazole, followed by deprotection; D: N-1-tritylsulfenyl-histamine (3) +
isocyanate, followed by deprotection; E: N-1-tritylsulfenyl-histamine (3) + isothiocyanate, followed by deprotection; F: N-1-tritylsulfenyl-histamine (3) +
cyanamide, followed by deprotection; G: N-1-tritylsulfenyl-histamine (3) + dicyandiamide, followed by deprotection.

36
Substitution patterns leading to efficient CA activators
and thioureido compounds were much less active,
whereas the guanidino/bis-guanide ones were more active
against all three investigated isozymes. Generally, no
great differences in activity were evidenced between the
isocyanates, the isothiocyanates or the guanidines of this
type, although the sub-series is relatively small for the
moment.
were: (i) perfluoroalkyl and perfluoroaryl, such as in
derivatives A1, A2, or A20, which were among the most
active compounds in the whole series of derivatives A,
against all three isozymes; (ii) For the substituted-aryl
derivatives, good CA activation was detected for com-
pounds possessing 4-halogenophenyl; 4-, 3- or 2-
iodophenyl, 4-, 3- or 2-toluenesulfonylureido-phenyl as
well as 2-, 3- or 4-pyridyl- moieties in their molecule; (iii)
very efficient activators were also those derived from
polyamino-polycarboxylic acids, such as nitrilotriacetic
(NTA); EDTA (ethylenediaminotetraacetic acid) or DTPA
(diethylenetriaminopentaacetic acid), both in their mono-
as well as bis-derivatives, of types A27–A29 or C1 and
C2. The many heteroatoms present in these moieties
probably contribute to the obtained compounds ‘sticky’
properties, i.e., they are able to participate in many
interactions with amino acid residues from the active site,
thus assuring the formation of very stable E–A (enzyme-
activator) adducts; (iv) An interesting case is represented
by the two compounds containing free sulfamoyl moi-
eties in their molecule, A36 and A38, which behave as
very strong inhibitors of all the investigated isozymes.
These two compounds, and the structurally related
probenecid derivative A37 were designed in such a way
as to contain a moiety able to interact with the inhibitor
binding site of the enzyme (i.e., the Zn(II) ion, where the
sulfonamide anion coordinates) and with the activator
binding site, where the histamine molecule binds (the
edge of the entrance to the cavity, between amino acid
residues Asn 62, His 64, and Gln 92 (figure 1). It was
important to understand which of the binding forces
would be stronger, and it is obvious from the data of
table I that the inhibitory one predominates. It is also
probable that the very strong inhibition observed with
these two compounds (A36 and A38) might be due to a
binding in which the activator-binding site also partici-
pates in the interaction, further stabilizing the E–I adduct.
The probenecid derivative A37 on the other hand behaves
as a relatively potent CA activator, since its sulfonamido
moiety, being substituted, is unable to interact with the
metal ion; (v) Slightly less active compounds were those
containing long alkyl chains, carboxy-alkyl and carboxy-
alkenyl ones, such as in compounds A5–A9; (vi) The
ureido/thioureido derivatives of type B40–B49 included
some of the best CA activators reported here, with
affinities in the 3–6 nM range for hCA I, 80–150 nM for
hCA II and 10–30 nM for bCA IV (table I). The
halogeno-containing derivatives B40–B43 were slightly
more active than the 1-naphthyl- or allyl derivatives B44
and B45, but for the moment this subseries of compounds
is too small for having a detailed SAR. The simple ureido
5. Conclusions
Similarly to all CA activators reported up to now, the
compounds obtained in the present work presumably
intervene in the catalytic cycle, leading to the formation
of an enzyme–activator complex (similarly to the en-
zyme–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 ca-
talysis) [7–13]. The driving force of this effect might be
the fact that intramolecular reactions are more rapid than
intermolecular ones. Thus, in the presence of activators
(symbolized as ‘A’), the rate-limiting step is described by
equation 1 below [7–13]:
EZn2+– OH2 + A
[EZn2+– OH2 – A]
EZn2+–OH– + AH+ (1)
[EZn2+–HO– – AH+]
enzyme–activator complexes
Obviously, compounds of the types reported here
possess the imidazolic moiety which can participate in
the proton transfer processes between the active site and
the environment (similarly to histamine 1), but due to the
presence of alkyl/arylcarboxamido (ureido) tails in their
molecule they can bind more effectively to the enzyme,
thus allowing for more efficient activation processes as
compared to 1. Indeed, the active site edge of all three CA
isozymes investigated by us contain a high proportion of
polar amino acid residues which might interfere with
polar groups such as RCONH or RNHCXNH (X = O, S,
NH). In fact, such amino acid residues might explain the
different catalytic properties of the diverse isozymes, as
well as their diverse susceptibility to be inhibited/
activated by modulators 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
different conformations (as shown by X-ray crystallogra-
phy) [7, 8] which could easily participate in hydrogen
bond formation (as well as other types of interactions)
with the histamine derivatives reported here. This might
explain, in fact, the greater efficiency of the compounds

37
reported in the present work in activating this isozyme, as
compared to histamine, which is a relatively weak hCA II
activator.
containing 3 was brought to pH 7 with solid NaHCO3,
brought to a small volume by in vacuo evaporation of the
solvent, and the precipitated 3 was then recrystallized
from ethanol (yield of 80%, based on histamine, after the
three steps described above). Tan crystals, m.p.
6. Experimental protocols
1
177–178 °C, H-NMR (300 MHz, DMSO-d6), δ 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, imida-
zole CH); Anal. (C24H23N3S) C, H, N, S.
6.1. Chemistry
Melting points were determined with a heating plate
microscope and are not corrected; IR spectra were ob-
tained in KBr pellets with a Perkin-Elmer 16PC FTIR
1
spectrometer, whereas H-NMR spectra with a Varian
6.1.2. General procedure for the preparation of
300CXP apparatus in solvents specified in each case.
Chemical shifts are expressed as δ 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 HPLC was done (C18 reversed-phase Bonda-
pack or Dynamax-60A (25 × 250 mm) columns.
Compounds used in synthesis (histamine, carboxylic
acids, carboxylic acid anhydrides, acyl chlorides, alkyl/
aryl isocyanates/isothiocyanates, cyanamide, dicyandia-
mide, tritylsulfenyl chloride, tetrabromophthalic anhy-
dride, hydrazine, EDTA dianhydride, DTPA dianhydride,
etc.) were commercially available compounds (from
Sigma, Acros or Aldrich). The tosylureido-amino acid
derivatives were prepared as described previously [23] by
the reaction of tosylisocyanate with amino acids. Aceto-
nitrile, dioxane (Merck) or other solvents used in the
synthesis were doubly distilled and kept on molecular
sieves in order to maintain them in anhydrous conditions.
compounds A1–A39 and B40–B49
Methods A–C: an amount of 10 mM N-1-trityl-
sulfenyl-histamine 3 was dissolved in 50 mL of anhy-
drous acetonitrile and then treated with a solution ob-
tained from 10 mM of carboxylic acid anhydride (method
A); acyl chloride (10 mM) and Et3N (10 mM) (method B)
or carboxylic acid (10 mM), diisopropyl-carbodiimide (or
EDCI) (10 mM) and 1-hydroxybenzotriazole (10 mM)
(method C) in the same solvent. The reaction mixture was
stirred at 4 °C 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 filtered, and the solvent removed in vacuo. In many
cases the compounds of type 4 precipitated, were filtered,
dried and deprotected at the N-1 imidazolic moiety in the
following way. The crude 4 was dissolved in 20 mL of
dioxane and treated with 25 mL of a 4 M HCl solution in
dioxane, followed by heating at reflux for 2 h. The
solvent was evaporated, the residue taken up in 50 mL of
a 5% solution of sodium bicarbonate and the trityl
sulfenyl chloride formed during the deprotection step
extracted in 2 × 50 mL of Et2O. The water phase was
evaporated in vacuo to a small volume, at which point,
generally, compounds A1–A39 precipitated by letting the
mixture stand at 4 °C overnight. The pure compounds
were obtained after recrystallization from ethanol/water
(1:1, v/v). In some cases, preparative HPLC was done
(C18 reversed-phase Bondapack or Dynamax-60A (25 ×
250 mm) columns; 90% acetonitrile/8% methanol/2%
water, 30 mL/min) in order to obtain the pure compounds
of type A1–A39.
6.1.1. Preparation of N-1-tritylsulfenyl-histamine 3
An amount of 5.55 g (50 mM) of histamine and
23.15 g (50 mM) of tetrabromophthalic anhydride were
suspended in 300 mL of dry toluene and refluxed under
Dean-Stark conditions until water was separated (gener-
ally 2–3 h). The solvent was evaporated in vacuo, the
crude product dissolved in 150 mL of anhydrous aceto-
nitrile and was treated with 15.5 g (50 mM) of tritylsulfe-
nyl chloride and 6.95 mL (50 mM) of triethylamine. The
mixture was stirred at room temperature for 3 h (TLC
control), then the solvent was evaporated and the crude
product stirred with 100 mL of water and ice. The tan
precipitate obtained was filtered, dried and used directly
in the deprotection step. Hydrazinolysis was effected by
dissolving the above mentioned precipitate in 200 mL of
ethanol and addition of 15 mL of hydrazinium hydroxide,
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 tetrabromophthalhy-
drazide was filtered and discarded, whereas the solution
Methods D and E: 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 isocyanate (method D) or
isothiocyanate (method E) dissolved in the same solvent.
The mixture was refluxed for 2–4 h, then the work-up and

38
the deprotection was effected as described above in
methods A–C.
6.1.6. 14-(1H-Imidazole-4-yl)-3-[2-[[2-(1H-Imidazole-4-
yl)ethyl]amino]-2-oxoethyl]-6,9-bis(carboxymethyl)-11-
oxo-3,6,9,12-tetraazatetradecanoic acid C2
Methods F and G: an amount of 10 mM N-1-
tritylsulfenyl-histamine 3 was dissolved in 50 mL of
ethanol and then treated with a solution obtained from
12 mM of cyanamide (method F) or dicyandiamide
(method G), and 5 mL of concentrated aqueous HCl. The
reaction mixture was refluxed for 2 h, then the solvent
was evaporated in vacuo, the tritylsulfenyl chloride
extracted with Et2O, and the crude guanidines B48 and
B49 recrystallized from iso-propanol.
1
White crystals, m.p. 173–175 °C; H-NMR (DMSO-
d6), δ, ppm: 2.45 (t, 2H, J = 7.0 Hz, histamine CH2); 2.94
(t, 2H, J = 7.0 Hz, histamine CONHCH2); 3.19 (t, 4H,
ethylenic CH2 near lateral nitrogens); 3.35 (s, 4H, CH2 of
the lateral acetates); 3.47 (t, 4H, ethylenic CH2 near
central nitrogen); 3.56 (s, 4H, CH2 of the acetamido
groups); 3.89 (s, 2H, CH2 of the central acetate); 7.34 (m,
1H, imidazole CH); 8.35 (s, 1H, imidazole CH); 8.80 (s,
1H, imidazole NH); 13C-NMR (DMSO-d6), δ, ppm: 33.3;
37.9; 51.6; 54.4; 56.0; 59.8; 60.7; 135.3; 145.8; 154.9;
171.6 (central COOH); 173.6 (CONH); 180.1 (lateral
COOH); Anal. (C24H37N9O8) C, H, N.
6.1.3. Preparation of compounds C1 and C2
The procedure of Aimé et al. [26] (developed for
DTPA-based NMR contrast agents) has been followed,
since it was observed that no N-1 acylated derivatives
were formed, and protection of the imidazolic NH group
was thus not necessary. An amount of 222 mg (2 mM) of
histamine was dissolved in 20 mL of anhydrous acetoni-
trile and treated with 1 mM of EDTA dianhydride or
DTPA dianhydride dissolved in the same solvent. The
mixture was stirred at room temperature for 6 h, the
solvent was evaporated and the crude residue recrystal-
lized from acetone/ethanol (2:1). Yields in C1 and C2
were around 95%.
6.2. Pharmacology
Human CA I and CA II cDNAs were expressed in
Escherichia coli strain BL21 (DE3) from the plasmids
pACA/hCA I and pACA/hCA II described by Forsman et
al. [27] (the two plasmids were a gift from Prof. Sven
Lindskog, Umea University, Sweden). Cell growth con-
ditions were those described by Lindskog’s group [28],
and enzymes were purified by affinity chromatography
according to the method of Khalifah et al. [29]. Enzyme
concentrations were determined spectrophotometrically
6.1.4. 4-[(-(Pyridine-2-carboxamidoethyl)]-
1H-imidazole A24
at 280 nm, utilizing
a
molar absorptivity of
49 mM–1.cm–1 for hCA I and 54 mM–1.cm–1 for hCA II,
respectively, based on Mr = 28.85 kDa for hCA I, and
29.30 kDa for hCA II, respectively [30, 31]. bCA IV was
isolated from bovine lung microsomes as described by
Maren et al., and its concentration was determined by
titration with ethoxzolamide [32].
White crystals, m.p. > 300 °C; IR (KBr), cm–1: 1 295
(amide III), 1 565 (amide II), 1 690 (amide I), 3 060
(NH); 1H-NMR (DMSO-d6), δ, ppm: 2.47 (t, 2H, J = 7.0
Hz, CH2); 2.93 (t, 2H, J = 7.0 Hz, CONHCH2); 7.34 (m,
1H, imidazole CH); 7.62 (t, 1H, J = 7.9 Hz, H4 of
pyridine); 7.70–7.93 (m, 3H, H3, H5 and H6 of pyridine);
8.15 (s, 1H, CONH); 8.35 (s, 1H, imidazole CH); 8.80 (s,
1H, imidazole NH); 13C-NMR (DMSO-d6), δ, ppm: 33.3;
37.9; 121.5; 125.9; 137.2; 157.3; 164.4; 167.1; 176.9
(CONH); Anal. (C11H12N4O) C, H, N.
Initial rates of 4-nitrophenyl acetate hydrolysis cataly-
sed by different CA isozymes were monitored spectro-
photometrically, at 400 nm, with a Cary 3 instrument
interfaced with an IBM compatible PC [33]. Solutions of
substrate were prepared in anhydrous acetonitrile; the
substrate concentrations varied between 2 × 10–2 and
1.10–6 M, working at 25 °C. A molar absorption coeffi-
cient e of 18 400 M–1.cm–1 was used for the
4-nitrophenolate formed by hydrolysis, in the conditions
of the experiments (pH 7.40), as reported in the litera-
ture [34]. Non-enzymatic hydrolysis rates were always
subtracted from the observed rates. Duplicate experi-
ments were done for each activator concentration and the
values reported throughout the paper are the means of
such results. Stock solutions of activator (1 mM) were
prepared in distilled-deionized water with 10–20% (v/v)
DMSO and dilutions up to 0.01 nM were done thereafter
with distilled-deionized water. Activator and enzyme
6.1.5. 4-[(-(4-Chlorophenylureido)-ethyl)]-
1H-imidazole B40
White crystals, m.p. > 294 °C (dec.); IR (KBr), cm–1:
1 287 (amide III), 1 579 (amide II), 1 710 (amide I),
3 060 (NH); 1H-NMR (DMSO-d6), δ, ppm: 2.49 (t, 2H, J
= 7.0 Hz, CH2); 2.99 (t, 2H, J = 7.0 Hz, CONHCH2); 7.34
(m, 1H, imidazole CH); 7.44 (d, 2H, J = 7.8 Hz,
p-Cl-C6H4); 7.79 (d, 2H, J = 7.8 Hz, p-Cl-C6H4); 8.21 (br
s, 2H, NHCONH); 8.35 (s, 1H, imidazole CH); 8.80 (s,
1H, imidazole NH); 13C-NMR (DMSO-d6), δ, ppm: 33.3;
37.9; 130.5; 134.5; 135.3; 145.8; 154.9 (NHCONH);
157.3; 164.4; 167.1; Anal. (C12H13ClN4O) C, H, N.

39
[13] Ilies M.A., Banciu M.D., Ilies M., Chiraleu F., Briganti F., Scoz-
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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 activation constant
KA was determined as described in ref. [7]. 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 activity [34] and higher concentrations had to be
used for the measurements).
[14] Sly W.S., in: Dodgson S.J., Tashian R.E., Gros G., Carter N.D.
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[18] Wolin R., Connolly M., Afonso A., Hey J.A., She H., Rivelli M.A.,
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Acknowledgements
This research was financed by the EU grant ERB
CIPDCT 940051.
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