Invesgations on the Influence of Halide Substituents on the Estrogen Receptor Interaction of 2,4,5-Tris(4-hydroxyphenyl)imidazoles

Article abstract of DOI:10.1002/ardp.200300729

Previously, we reported on the synthesis and estrogen receptor (ER) interaction of imidazoles, which had to be 1-alkyl-4,5-bis(2-halo-4-hydroxyphenyl) substituted for a high relative binding affinity (RBA > 1 %). This led to the assumption that a shielding of the polar heterocyclic system is a prerequisite for ER binding. In continuation of this study we synthesized 2,4,5-tris(4-hydroxyphenyl)imidazoles with Cl- or F-atoms in the ortho-positions of the aromatic rings and evaluated whether they mediate sufficient hydrophobicity for ER interaction. 2-(2,6-Dichloro-3/4-hydroxyphenyl)-4,5-bis(2-halo-4-hydroxyphenyl)imidazoles were synthesized by reaction of the respective methoxy-substituted benzil with either the 2,6-dichloro-4-methoxy- or the 2,6-dichloro-3-methoxybenzaldehyde in ammonium acetate solution. The required ether cleavage was performed subsequently with BBr₃. In the competition experiment with [ ³H]estradiol the imidazoles with the a C2-standing (2,6-dichloro-4-hydroxyphenyl) ring showed an RBA > 0.02 %, but did not activate the luciferase gene in estrogen receptor positive MCF-7-2a breast cancer cells stably transfected with the plasmid ERE_wtcluc. In the test for antagonistic potency only the 2-(2,6-dichloro-4-hydroxyphenyl)-4,5-bis(4-hydroxyphenyl)imidazole 3 antagonized the effects of 1 nM estradiol slightly. From these data, it can be concluded that a C2-standing 2,6-dichloro-4-hydroxyphenyl ring is not appropriate to optimize the ER interaction of 4,5-(4-hydroxyphenyl)imidazoles.

Full text of DOI:10.1002/ardp.200300729

456 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
Ronald Gust,
Invesgations on the Influence of Halide
Substituents on the Estrogen Receptor Interaction
of 2,4,5-Tris(4-hydroxyphenyl)imidazoles
Sandra Busch,
Roland Keilitz,
Kathrin Schmidt,
Moriz von Rauch
Previously, we reported on the synthesis and estrogen receptor (ER) interaction of
imidazoles, which had to be 1-alkyl-4,5-bis(2-halo-4-hydroxyphenyl) substituted for
a high relative binding affinity (RBA > 1 %).This led to the assumption that a shield-
ing of the polar heterocyclic system is a prerequisite for ER binding. In continuation
of this study we synthesized 2,4,5-tris(4-hydroxyphenyl)imidazoles with Cl- or F-at-
oms in the ortho-positions of the aromatic rings and evaluated whether they mediate
sufficient hydrophobicity for ER interaction. 2-(2,6-Dichloro-3/4-hydroxyphenyl)-
4,5-bis(2-halo-4-hydroxyphenyl)imidazoles were synthesized by reaction of the re-
spective methoxy-substituted benzil with either the 2,6-dichloro-4-methoxy- or the
2,6-dichloro-3-methoxybenzaldehyde in ammonium acetate solution.The required
ether cleavage was performed subsequently with BBr3. In the competition experi-
ment with [3H]estradiol the imidazoles with the a C2-standing (2,6-dichloro-4-hy-
droxyphenyl) ring showed an RBA > 0.02 %, but did not activate the luciferase gene
in estrogen receptor positive MCF-7-2a breast cancer cells stably transfected with
the plasmid EREwtcluc.In the test for antagonistic potency only the 2-(2,6-dichloro-4-
hydroxyphenyl)-4,5-bis(4-hydroxyphenyl)imidazole 3 antagonized the effects of 1
nM estradiol slightly. From these data, it can be concluded that a C2-standing 2,6-
dichloro-4-hydroxyphenyl ring is not appropriate to optimize the ER interaction of
4,5-(4-hydroxyphenyl)imidazoles.
Institute of Pharmacy,
Free University of Berlin,
Königin-Luise Str. 2 + 4,
D-14195 Berlin, Germany
Keywords: Estrogenic activity; Luciferase assay; Estrogen receptor binding; 2,4,5-
triarylimidazoles
Received: September 24, 2002, Accepted: April 1, 2003 [FP729]
DOI 10.1002/ardp.200300729
E2 and DES are oriented in the LBD in such a way that
the OH groups build H-bridges to Glu353, Arg 394, and
Introduction
The estrogen receptor (ER) is a ligand- inducible nuclear
transcription factor for the mediation of the effects of es-
trogen steroid hormones [1]. Through binding to the lig-
and binding domain (LBD) of the ER the hormones initi-
ate in many tissues of the reproduction system a cas-
cade of molecular and biochemical effects [2]. Further-
more, the ER also plays an important role in bone densi-
ty maintenance [3–5], regulation of the blood/lipid profile
[4–8], and brain function [9, 10]. Therefore, many scien-
tific groups used the ER as a target for the discovery of
selectively acting new drugs (Scheme 1).
His 524. Lipophilic residues of amino acids in the LBD
are in van der Waals contacts with the hydrophobic core
of the molecules and stabilize the ER/drug conjugate.
4OHT and RAL occupy the same central core of the
LBD, and the basic side chains are oriented in a narrow
side pocket building an H-bridge to Asp351.This anchor-
age fixes RAL in a plane conformation.
The crystal structures confirm the assumption that espe-
cially flat and rigid molecules interact with the ER. Fol-
lowing this strategy, the carbon atoms of the ethyl group
of tamoxifen (TAM) were linked back to the proximate
phenyl ring to reduce the flexibility. Resulting diphenylin-
denes [14], diphenyldihydro- or diphenyltetrahydronaph-
thalene derivatives [15–17], or TAM analogs with larger
rings were subjected to intensive structure activity stud-
ies [18]. Further investigated heterocycles are benzo-
furans [19], benzothiophenes [20], benzopyrans [21],
and indoles [22]. In each case, the fused bicyclic system
is thought to mimic the A/B ring of E2.
The recently solved X-ray structures [11–13] of the LBD
of ERα co-crystallized with agonists (e.g., estradiol (E2),
diethylstilbestrol (DES)) and antagonists (e.g.,
raloxifene (RAL), 4-hydroxytamoxifen (4OHT) are help-
ful tools for the design of compounds with ER affinity.
Correspondence: Ronald Gust, Institut für Pharmazie der FU
Berlin, Königin Luise Str. 2+4, D-14195 Berlin, Germany.
Phone: +49-(030)-838-53272, Telefax: +49-(030)-838-56906,
e-mail: rgust@zedat.fu-berlin.de
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
Halide Substituents and Estrogen Receptor Interaction 457
Scheme 1. The structural formulae of compounds referred to in the text.
Rigid molecules with very interesting pharmacological
properties are tetrasubstituted pyrazoles, furans, thio-
phenes, and pyrroles developed by the group of J. A.
Katzenellenbogen [23, 24]. ER binding and transcrip-
tional activation studies showed that several members of
these series possess high ER subtype selectivity and
can be used as lead structures for the design of selective
estrogen receptor modulators (SERM).
phenyl)imidazole showed a relative binding affinity of
1.26 %. The significance of an alkyl substituent at the
1-position for ER binding and gene activation of 4,5-
bis(4-hydroxyphenyl)imidazoles was already confirmed
by Fink et al.[26].However, this effect depends on an ad-
ditional 4-OH-phenyl ring in the 2-position. The 2,4,5-
tris(4-hydroxyphenyl)-1-propylimidazole was identified
as most active compound with an RBA = 0.62 %.The au-
thors interpreted the relatively low binding affinity with
the high inherent polarity of the heterocyclic system and
the high dipole moment.
In our own studies, we selected the 1-alkyl-4,5-bis(2-ha-
lo-4-hydroxyphenyl)imidazole for the design of novel ER
agonists [25]. The 1-ethyl-4,5-bis(2-chloro-4-hydroxy
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

458 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
Scheme 2. Synthesis of the triarylimidazoles 1–5 (X = H, F, Cl; R = 2,6-Cl2,3-OH/3-OCH3; 2,6-Cl2,4-OH/4-OCH3).
In an earlier study, we evaluated the influence of halide
substituents in the aromatic rings of estrogenically active
1,2-bis(4-hydroxyphenyl)ethylenediamines, which were
designed as carrier molecules for metal complexes [27,
28].These compounds interact with the ER and exert es-
trogenic effects if at least one phenyl ring possesses a
2,6-dichloro-3/4-hydroxy substitution pattern. The or-
tho-standing substituents shield the hydrophilic amino
groups allowing hydrophobic contacts in the LBD of the
ER.These positive results prompted us to shield the imi-
dazole core by introduction of a 2,6-dichloro-3/4-hy-
droxyphenyl ring into the 2-position. The resulting 2-
(2,6-dichloro-3/4-hydroxyphenyl)-4,5-bis(2-halo-4-hy-
droxyphenyl)imidazoles were evaluated for ER binding
as well as for agonistic and antagonistic properties in
MCF-7-2a cells stably transfected with the plasmid
EREwtcluc.
densation of respectively substituted benzaldehydes
and oxidation with CuSO4 (Scheme 2).The subsequent
ring formation was performed with the 2,6-dichloro-3/4-
methoxybenzaldehydes 9 c and 9 d in a modified Rad-
ziszewski reaction [29–31]. Ether cleavage with BBr3
yielded the imidazoles 1–5.
The heterocyclic skeleton represents an aromatic sys-
tem in which the substituents at the C atoms are located
in the heterocyclic plane.Figure 1 shows the energetical-
ly preferred conformations of the 4,5-bis(2-chloro-4-hy-
droxyphenyl)-2-(2,6-dichloro-4-hydroxyphenyl)imid-
azole 5 and the 4,5-bis(2-chloro-4-hydroxyphenyl)imid-
azole DPI.The arrangement of the aryl rings in 4,5-posi-
tion (diarylethene pharmacophor) is independent of the
C2 aryl substituent. The torsion angles amount to 6.9°
(5) and 5.8° (DPI) and the distance of the OH groups is
nearly identical (8.8 and 8.9 Å).The 4-OH group in the 2-
phenyl ring of 5 is arranged in a distances of 12.9 Å to
those in the 4,5-standing rings.
Results
Synthesis and structural characterization
1H-NMR spectroscopic studies revealed that the NH-
proton at the heterocyclic ring of 5 is fixed in DMSO solu-
tion.In contrast, the heating of a DMSO solution of DPI to
The synthesis of 2,4,5-triarylimidazoles started from
benzils (6, 7, 8), which can be obtained by benzoin con-
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Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
Halide Substituents and Estrogen Receptor Interaction 459
Figure 1. Comparison of the spatial structures of the 4,5-bis(2-chloro-4-hydroxyphenyl)imidazole DPI (left) and 4,5-
bis(2-chloro-4-hydroxyphenyl)-2-(2,6-dichloro-4-hydroxyphenyl)imidazole 5 (right).
Table 1. Relative binding affinity and agonistic/antagonistic effects of the 2,4,5-triarylimidazoles 1 to 5 in the luciferase
assay.
Rel. activation of luciferase
Inhibition [%] of the
expression [% of E2]
E2 effect (1 nM)
at 10–6
M
at 10–6
M
X
R
RBAa
[%]
–OH
derivatives
–OCH3
derivatives
–OH
derivatives
1
2
3
4
5
H
H
H
F
4-OH
<0.02
<0.02
0.02
0.03
0.19
0.11
3
2
3
3
7
8
5
8
5
0
1
14
13
28
2,6-Cl2, 3-OH
2,6-Cl2, 4-OH
2,6-Cl2, 4-OH
2,6-Cl2, 4-OH
22
Cl
15
DPI
–3
n.d.b
a
b
Relative binding affinity (RBA), % = [E2]/[I] × 100;[E2] and [I] are the molar concentrations of nonradioactive E2 and in-
hibitor required to decrease the bound radioactivity by 50 %; E2 = 17β-estradiol. Mean value of 3 determinations.
Not determined.
413 K led to proton exchange reactions and therefore to
an equivalence of the nitrogens. This effect was ob-
served for 5 only in acidic solution.
E2 and the used inhibitor required to reduce the receptor
binding of [3H]-E2 to 50 %.
As listed in Table 1 only the imidazoles 3, 4, and 5 com-
peted with E2 for its binding site.The RBA = 0.19 % of the
most active compound, the 4,5-bis(2-chloro-4-hydroxy-
Biological activity
The affinity to the binding site of E2 was quantified in a
competition experiment using calf uteri cytosol and [3H]-
E2 [32].The relative binding affinity (RBA, E2 = 100 %) is
the proportion of the molar concentrations of unlabeled
phenyl)-2-(2,6-dichloro-4-hydroxyphenyl)imidazole
5
showed that a 2-standing 2,6-dichloro-4-hydroxyphenyl
ring increased the ER binding only marginally (DPI:RBA
= 0.11 %).
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460 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
The gene activation resulting from the ER binding was
evaluated concentration dependent (10–10 M to 10–5 M)
in a luciferase assay using MCF-7-2a cells.The ER-posi-
tive human breast cancer cells are stably transfected
with the reporter plasmid EREwtcluc. The binding of ER/
drug dimers at the estrogen response elements (ERE) of
the plasmid activates the luciferase gene.The quantifica-
tion of the luciferase expression allows a prediction of the
agonistic potency, the inhibition of the E2 induced activa-
tion correlates with the antagonistic effects of the com-
pounds [33].
The effects on the MCF-7-2a cell line were low. None of
the compounds activated the gene expression despite
the enhanced hydrophobicity due to the 2-standing 2,6-
dichloro-3/4-hydroxy substituted phenyl ring. The E2
(1 nM) stimulated luciferase expression was only re-
duced significantly by 3 in the concentrations of 10–6
M
(28 %) and 10–5 M (38 %).
Discussion
Figure 2. Superposition of the 4,5-bis(2-chloro-4-
hydroxyphenyl)-2-(2,6-dichloro-4-hydroxyphenyl)imid-
azole 5 and raloxifene in the LBD of the estrogen recep-
tor.
Planar heterocyclic rings are well known as potential
lead structures for the design of SERM. Especially imid-
azoles, pyrazoles and related compounds were subjects
of structure activity studies [23, 14, 26, 34].Fink et al.[26]
investigated the ER-interaction of the 4,5-bis(4-hydroxy-
phenyl)imidazole (RBA < 0.001 %) and showed that a
third phenol ring in position 2 (RBA = 0.007 %) and an
additional ethyl chain at the N(1) atom (RBA = 0.38 %)
increased the relative binding affinity.
hance the gene expression. It is assumed that, due to
this anchorage, the type II estrogens do not activate the
activation function AF2, but use the novel AF2b site [36]
that includes acidic surface amino acids on a reposi-
tioned helix 12, an exposed aspartate at position 351
and AF1.
In our studies, we demonstrated [25] that 4,5-bis(4-hy-
droxyphenyl)imidazoles bind to the ER with high affinity,
if the ortho-positions of the aromatic rings are Cl-substi-
tuted. In this series, the 1-ethyl-4,5-bis(2-chloro-4-
hydroxyphenyl)imidazole possessed the highest RBA
with 1.26 %. O-methylation prevented the ER binding.
This indicates that 4,5-diarylimidazoles are attached to
the LBD analogously to E2 or DES by a combination of
hydrophobic contacts and H-bridges.
In this structure activity study, we tried to optimize the ER
interaction of 4,5-bis(4-hydroxyphenyl)imidazoles by in-
troduction of a 2,6-dichloro-4-hydroxyphenyl or 2,6-di-
chloro-3-hydroxyphenyl ring into the 2-position of the
heterocyclic ring. In analogy to 1,2-diarylethylenedi-
amines [27, 28], this substitution pattern guarantees the
shielding of the electron rich imidazole core by the ortho-
Cl substituents and the contact to His524. As demon-
strated in Figure 1, the distances between the hydroxy
groups located in the 4,5- and the 2,4/2,5-standing aro-
matic rings amount to 8.8 Å and 12.9 Å, respectively.The
latter is nearly identical to that of DES.
The 1,2-diarylethene pharmacophor of the 4,5-diarylimi-
dazoles is arranged in a Z-stilbene-like structure. An
anchorage in the LBD comparable to DES or E2 is im-
possible. Therefore, the hypothetical binding model for
type II estrogens [35] must be used for 4,5-diarylimid-
azoles, since no crystal structures of the LBD occupied
with to these compounds are available.
Two orientations are possible in the LBD due to the sym-
metric character of the molecules.The first one based on
our model for the ER binding of type II estrogens at which
the 2,6-dichloro-4-hydroxy-substituted aromatic ring
contacts His524 and the 4,5-diarylimidazole moiety is at-
tached to Glu353, Arg394, and Asp351.This orientation
correlates very well with that of RAL (Figure 2).
In contrast to type I estrogens (E2, DES, etc.), which
contact His524, type II estrogens of the piperazine or im-
idazoline series seem to be attached to Asp351 in the hy-
drophobic side pocket. Lipophilic parts of the molecules,
especially ortho-Cl substituents are in van der Waals
contacts with lipophilic residues of amino acids and en-
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Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
Halide Substituents and Estrogen Receptor Interaction 461
Fink et al. [26] investigated in a structure/activity study
novel templates for ER ligands and prepared several
triphenol imidazole and pyrazole derivatives. They
showed that compounds of both classes interact with the
ER and postulated an orientation in the LBD comparable
to our model.
Experimental
General procedures
IR spectra (KBr pellets): Perkin Elmer Model 580 A (Shelton,
USA). 1H-NMR: Bruker ADX 400 spectrometer at 400 MHz (in-
ternal standard: TMS) (Rheinstetten, Germany). Elemental
analyses: Microlaboratory of Free University of Berlin.
Recently, a second binding mode was suggested for
tris(4-hydroxyphenyl)-substituted five membered hete-
rocycles. 3-Ethyl-2,4,5-tris(4-hydroxyphenyl)furan (TPF,
Scheme 1) and 4-propyl-1,3,5-tris(4-hydroxyphenyl)-
pyrazole (TPP, Scheme 1) are oriented in the LBD in
such a way that the 2,4-bis(4-hydroxyphenyl)furan or the
3,5-bis(4-hydroxyphenyl)pyrazole moiety mimics the
E-stilbene partial structure of DES as well as the steroi-
dal skeleton of E2.In this case the N(1)- or C(5)-standing
phenol ring cannot be oriented in the narrow side pocket,
but can make a hydrogen bond with the OH group of
Thr347, which is the only polar residue in the ligand bind-
ing pocket.This binding mode was made responsible for
high affinity selectivity for ERα, which increases 408
times for TPP and 48 times for TPF.
EI-MS-spectra: CH-7A-Varian MAT (70eV) (Palo Alto, USA) or
Kratos MS 25 RF (80eV) (New York, USA). All computational
graphics were built using SYBYL 6.6, Tripos Inc., (1699 South
Hanley Rd., St. Louis, Missouri, 63144, USA). Geometry opti-
mization was carried out using the Tripos force field from within
the SYBYL program, running on an INDY workstation. Liquid
Scintillation Counter: 1450 MicrobetaTM Plus (Wallac, Turku,
Finland).Microlumat:LB 96 P (BertholdTechnologies, BadWil-
dungen, Germany).
Syntheses
The benzaldehydes 9 a to 9 d were synthesized as described
earlier [28], 4,4Ј-dimethoxybenzil and 4-methoxybenzalde-
hyde are commercially available.
General procedure for the synthesis of 2,2Ј-dihalo-4,4Ј-dimeth-
oxybenzoines
A solution of 2-halo-4-methoxybenzaldehyde (20.0 mmol) and
KCN (5.00 mmol) in 15 mL of ethanol/water (60 %) was heated
to reflux for 12 h.Further 5.00 mmol KCN were added after 3, 6,
and 9 h. After the reaction completed, the solution was allowed
to cool to room temperature (RT). 30 mL of water were added
and it was extracted with CH2Cl2. The organic layer was
separated, washed 3 times with water, dried over Na2SO4, and
evaporated. The crude product was purified by chromatogra-
phy on silica gel with diethyl ether/ligroine (1 + 3).
Generally, the imidazoles 1–5 can be attached in the
same way in the LBD. If the contact to Thr347 plays an
important role in gene activation, it would cause effects
in the luciferase assay on MCF-7-2a cells, because
these are hormone dependent breast cancer cells with
an ERα content of about 95 %.
However, the imidazoles 1 to 5 possessed no agonistic
2,2Ј-Difluoro-4,4Ј-dimethoxybenzoin (6 a)
properties, and antagonistic effects were determined
From 2-fluoro-4-methoxybenzaldehyde 9 a: 8.43 mmol (1.30
g).Yield:0.811 mmol (250 mg), 19 %;pale yellow oil.IR (KBr):ν
= 3456 m, br (OH);2842 w (OCH3);1672 m (C=O);1616 s;1508
s; 1444 m; 1271 s; 1158 s; 1121 m; 1031 m; 978 m; 839 m; 756
s.1H-NMR (CDCl3):δ = 3.74 (s, 3 H, OCH3);3.81 (s, 3 H, OCH3);
4.50 (s, 1 H, OH, exchangeable by D2O); 5.97 (s, 1 H, ArCH);
6.49 (dd, 3J(H, F) = 13.0 Hz, 4J = 2.5 Hz, 1 H, ArH-3); 6.56 (dd,
3J(H, F) = 11.9 Hz, 4J = 2.4 Hz, 1 H, ArЈH-3); 6.60 (dd, 3J = 8.5
Hz, 4J = 2.5 Hz, 1 H, ArH-5); 6.73 (dd, 3J = 8.7 Hz, 4J = 2.4 Hz,
1 H, ArЈH-5); 7.07 (dd, 3J = 8.5 Hz, 4J(H, F) = 8.4 Hz, 1 H, ArH-
only for 3 in concentrations of 10–6 M (28 %) and 10–5
M
(38 %).
Conclusion
In this structure activity relationship study we showed
that ortho-Cl substituents in the C2-standing phenyl ring
are not appropriate to optimize the ER interaction of
2,4,5-tris(4-hydroxyphenyl)imidazoles causing gene ac-
tivation. Nevertheless, due to its low but significant an-
tagonistic profile in MCF-7-2a cells, 3 represents an
interesting lead structure for the design of SERM with
antagonistic effects in tumor cells.Therefore, we focused
our attention on the effects of alkyl substituents on the
hormonal properties of tris(4-hydroxyphenyl)imid-
azoles.
3
4
6); 7.92 (dd, J = 8.7 Hz, J(H, F) = 8.5 Hz, 1 H, ArЈH-6).
2,2Ј-Dichloro-4,4Ј-dimethoxybenzoin (7 a)
From 2-chloro-4-methoxybenzaldehyde 9 b: 11.7 mmol
(2.00 g). Yield: 0.909 mmol (310 mg), 16 %; colorless oil. IR
(Film): ν = 3467 m, br (OH); 2840 w (OCH3); 1688 m (C=O);
1601 s; 1497 s; 1239 s; 1049 s; 975 m; 758 s. 1H-NMR (CDCl3):
δ = 3.75 (s, 3 H, OCH3); 3.80 (s, 3 H, OCH3); 4.44 (s, 1 H, OH,
austauschbar exchangeable by D2O); 6.27 (s, 1 H, ArCH);
4
6.72–6.77 (m, 2 H, ArH-5); 6.84 (d, J = 2.4 Hz, 1 H, ArH-3);
6.88 (d, 4J = 2.3 Hz, 1 H, ArH-3); 7.10 (d, 3J = 8.6 Hz, 1 H, ArH-
3
6); 7.47 (d, J = 8.7 Hz, 1 H, ArH-6).
General procedure for the synthesis of 2,2Ј-dihalo-4,4Ј-di-
methoxybenzil
Acknowledgements
CuSO4 × 5 H2O (10.3 mmol) was dissolved in a mixture of
15.5 mL pyridine and 12.7 mL water. To the blue solution
2.50 mmol of the 2,2Ј-dihalo-4,4Ј-dimethoxybenzoine were
added and the reaction mixture was heated to 100 °C for 4 h.
Subsequently, the green mixture was cooled to RT, poured on
The technical assistance of S. Bergmann and I.
Schnautz is acknowledged. The presented study was
supported by Grant Gu285/3-1 from the Deutsche For-
schungsgemeinschaft.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

462 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
20 mL of water and extracted with CH2Cl2. The organic layer
was washed twice with 10 mL of 2 N HCl and 20 mL of water.
After drying over Na2SO4 the solvent was evaporated. The
crude product was purified by chromatography on silica gel
with diethyl ether/ligroine (1 + 1).
4,5-Bis(4-methoxyphenyl)-2-(2,6-dichloro-4-methoxyphenyl)-
imidazole (3 a)
From 4,4Ј-dimethoxybenzil (1.85 mmol (500 mg)) and 2,6-
dichloro-4-methoxybenzaldehyde 9 d (1.87 mmol (383 mg)).
Purification by re-crystallization from CHCl3.Yield: 0.988 mmol
(450 mg), 53 %;, colorless powder, mp.: 248–249 °C. IR (KBr):
ν= 3382 m, br (NH); 2936 m; 2835 m (OCH3); 1612 s; 1553 s;
1519 s;1495 s;1463 s;1294 s;1247 s;1177 s;1061 m;1037 s;
971 m; 900 m; 833 s; 804 m. 1H-NMR (CDCl3): δ = 3.80 (s, 3 H,
OCH3); 3.83 (s, 3 H, OCH3); 3.84 (s, 3 H, OCH3); 6.82 (AAЈBBЈ,
3J = 8.9 Hz, 2 H, ArH-3, ArH-5); 6.90 (AAЈBBЈ, 3J = 8.7 Hz, 2 H,
2,2Ј-Difluoro-4,4Ј-dimethoxybenzil (6)
From 2,2Ј-difluoro-4,4Ј-dimethoxybenzoin 6 a: 0.811 mmol
(250 mg).Yield: 0.754 mmol (231 mg), 93 %; pale yellow oil. IR
(KBr):ν = 2855 w (OCH3);1665 m (C=O);1616 s;1505 w;1444
m;1282 m;1244 m;1157 m;1099 m;1012 w;951 w;846 w. 1H-
NMR (CDCl3): δ = 3.88 (s, 6 H, OCH3); 6.60 (dd, 3J(H, F) =
3
ArH-3, ArH-5); 6.95 (s, 2 H, ArЈH); 7.38 (AAЈBBЈ, J = 8.7 Hz,
2 H, ArH-2, ArH-6); 7.57 (AAЈBBЈ, 3J = 8.9 Hz, 2 H, ArH-2, ArH-
6); 9.11 (s, 1 H, NH, exchangeable by D2O).
4
3
4
12.6 Hz, J = 2.3 Hz, 2 H, ArH-3); 6.85 (dd, J = 8.4 Hz, J =
2.3 Hz, 2 H, ArH-5);8.02 (dd, 3J = 8.4 Hz, 4J(H, F) = 8.0 Hz, 2 H,
ArH-6).
4,5-Bis(2-fluoro-4-methoxyphenyl)-2-(2,6-dichloro-4-meth-
oxyphenyl)imidazole (4 a)
2,2Ј-Dichloro-4,4Ј-dimethoxybenzil (7)
From 2,2Ј-difluoro-4,4Ј-dimethoxybenzil
6
(0.408 mmol
(125 mg)) and 2,6-dichloro-4-methoxybenzaldehyde 9 d
(0.410 mmol (84 mg)). Purification by re-crystallization from
CHCl3. Yield: 0.342 mmol (168 mg), 84 %; colorless powder,
mp.: 234–236 °C. IR (KBr): ν = 2942 w; 2840 w (OCH3); 1629 s;
1594 s;1553 m;1518 m;1491 s;1464 m;1438 m;1318 m;1287
s;1242 m;1193 m;1156 s;1069 m;1034 m;945 m;837 m;807
m. 1H-NMR (CDCl3): δ = 3.81 (s, 6H, OCH3); 3.85 (s, 3 H,
OCH3); 6.63 (dd, 3J(H, F) = 12.4 Hz, 4J = 2.1 Hz, 2 H, ArH-3);
From 2,2Ј-dichloro-4,4Ј-dimethoxybenzoin 7 a: 0.909 mmol
(310 mg).Yield:0.762 mmol (258 mg), 84 %;yellow oil.IR (KBr):
ν = 2842 w (OCH3); 1663 s (C=O); 1593 s; 1491 s; 1459 m;
1409 m;1304 s;1283 s;1238 s;1185 m;1145 w;1044 s;895 m;
860 m; 806 m; 760 s; 689 m. 1H-NMR (CDCl3): δ = 3.89 (s, 6 H,
OCH3); 6.92–6.97 (m, 4 H, ArH-3, ArH-5); 8.06 (d, 3J = 8.4 Hz,
2 H, ArH-6).
3
4
6.67 (dd, J = 8.6 Hz, J = 2.1 Hz, 2 H, ArH-5); 6.94 (s, 2 H,
ArЈH); 7.40 (br, 2 H, ArH-6).
General procedure for the synthesis of 2,4,5-triarylimidazole
A solution of the benzil (2.00 mmol), the benzaldehyde
(2.00 mmol ), and ammonium acetate (16.4 mmol, 1.26 g) in
10 mL of acetic acid was heated to reflux for 12 h. After evapo-
ration of the solvents, 15 mL of water were added and the prod-
uct was extracted with CH2Cl2. The organic layer was washed
with Na2CO3 solution (5 %) and water and was evapoprated af-
ter drying over Na2SO4.
4,5-Bis(2-chloro-4-methoxyphenyl)-2-(2,6-dichloro-4-meth-
oxyphenyl)imidazole (5 a)
From 2,2Ј-dichloro-4,4Ј-dimethoxybenzil
7 (0.719 mmol
(244 mg)) and 2,6-dichloro-4-methoxybenzaldehyde 9 d
(0.741 mmol (152 mg)). Purification was performed by column
chromatography on silica gel with diethyl ether and ligroine (3 +
1). Yield: 0.610 mmol (320 mg), 85 %; colorless powder, mp.:
189–191 °C.IR (KBr):ν = 2940 w;2840 w (OCH3);1610 s;1555
m; 1508 m; 1481 s; 1436 m; 1288 s; 1227 s; 1186 w; 1060 m;
1038 s; 842 m; 809 m. MS (EI, 220 °C): m/z (%) = 522 (82)
2,4,5-Tris(4-methoxyphenyl)imidazole (1 a)
From 4,4Ј-dimethoxybenzil (1.85 mmol (500 mg)) and 4-meth-
oxybenzaldehyde (1.90 mmol (259 mg)). For purification the
crude product was recrystallized from ethanol/ligroine. Yield:
0.983 mmol (380 mg), 53 %; colorless powder, mp.: 248–
249 °C. IR (KBr): = 3420 m, br (NH); 2998 w; 2838 m (OCH3);
1617 m;1520 s;1502 s;1293 m;1247 s;1177 s;1032 s;970 m;
833 s. 1H-NMR (CDCl3): δ = 3.81 (s, 6 H, OCH3); 3.83 (s, 3 H,
OCH3); 6.86 (AAЈBBЈ, 3J = 7.0 Hz, 4 H, ArH-3, ArH-5); 6.93
(AAЈBBЈ, 3J = 7.8 Hz, 2 H, ArЈH-3, ArЈH-5); 7.44 (AAЈBBЈ, 3J =
7.0 Hz, 4 H, ArH-2, ArH-6);7.80 (AAЈBBЈ, 3J = 7.8 Hz, 2 H, ArЈH-
2, ArЈH-6).
çs
[M]+ ; 507 (6) [M-CH3]+; 153 (15). 1H-NMR (CDCl3): δ = 3.79 (s,
3
4
6 H, OCH3); 3.86 (s, 3 H, OCH3); 6.72 (dd, J = 8.7 Hz, J =
2.3 Hz, 2 H, ArH-5); 6.89 (d, 4J = 2.3 Hz, 2 H, ArH-3); 6.92 (s,
3
2 H, ArЈH); 7.20 (d, J = 8.7 Hz, 2 H, ArH-6).
General procedure for the ether cleavage with BBr3
A solution of the methyl ether (1.00 mmol) in 20 mL of dry
CH2Cl2 was cooled to –60 °C. BBr3 (4.5 mmol) in 5 mL of dry
CH2Cl2 was added at this temperature under N2 atmosphere.
Then the reaction mixture was allowed to warm to room tem-
perature and was stirred for further 48 h.After cooling the reac-
tion mixture with an ice bath, the surplus of BBr3 was hydrolized
three times with methanol and the phenolic product was dis-
solved in 0.1 N NaOH. The alkaline/water phase was filtrated
and the pH was adjusted to 8 with 2 N HCl.The precipitate was
collected by suction filtration and dried over P2O5.Subsequent-
ly, the crude product was purified if necessary by column chro-
matography or fractional crystallization.
4,5-Bis(4-methoxyphenyl)-2-(2,6-dichloro-3-methox-
yphenyl)imidazole (2 a)
From 4,4Ј-dimethoxybenzil (1.85 mmol (500 mg)) and 2,6-
dichloro-3-methoxybenzaldehyde 9 c (1.87 mmol (383 mg)).
Purification was performed by column chromatography on sili-
ca gel with diethyl ether and ligroine (1 + 1) and subsequent re-
crystallization from isopropanol/diisopropyl ether. Yield:
1.10 mmol (500 mg), 59 %;, colorless powder, mp.: 126–
127 °C. IR (KBr): ν = 3382 m, br (NH); 2936 m; 2834 m (OCH3);
1615 m;1567 m;1518 s;1491 s;1459 s;1294 s;1249 s;1177 s;
1060 m; 1032 m; 834 s; 803 m. 1H-NMR (CDCl3): d = 3.82 (s,
6 H, OCH3);3.93 (s, 3 H, OCH3);6.87 (AAЈBBЈ, 3J = 8.2 Hz, 4 H,
ArH-3, ArH-5); 6.96 (d, 3J = 8.9 Hz, 1 H, ArЈH-4); 7.35 (d, 3J =
8.9 Hz, 1 H, ArЈH-5); 7.50 (br, 4 H, ArH-2, ArH-6); 9.15 (s, 1 H,
NH, exchangeable by D2O).
2,4,5-Tris(4-hydroxyphenyl)imidazole (1)
From 2,4,5-tris(4-methoxyphenyl)imidazole 1 a (0.621 mmol
(240 mg)).Purification was performed by re-crystallization from
isopropanol and diisopropyl ether.Yield: 0.569 mmol (196 mg),
92 %; pale grey powder, mp.: 195–198 °C. IR (KBr): ν = 3600–
2700 s, br (OH); 2970 w; 1648 m; 1615 s; 1503 s; 1380 m;1248
s; 1173 s; 1103 m; 836 s. 1H-NMR ([D6]-DMSO): δ = 6.67
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
Halide Substituents and Estrogen Receptor Interaction 463
3
3
(AAЈBBЈ, J = 8.5 Hz, 2 H, ArH-3, ArH-5); 6.79 (AAЈBBЈ, J =
8.4 Hz, 2 H, ArH-3, ArH-5);6.82 (AAЈBBЈ, 3J = 8.7 Hz, 2 H, ArH-
3, ArH-5); 7.26 (AAЈBBЈ, 3J = 8.4 Hz, 2 H, ArH-2, ArH-6); 7.33
4,5-Bis(2-chloro-4-hydroxyphenyl)-2-(2,6-dichloro-4-hy-
droxyphenyl)imidazole (5)
From
4,5-bis(2-chloro-4-methoxyphenyl)-2-(2,6-dichloro-4-
3
3
(AAЈBBЈ, J = 8.5 Hz, 2 H, ArH-2, ArH-6); 7.84 (AAЈBBЈ, J =
8.7 Hz, 2 H, ArH-2, ArH-6); 9.28 (s, 1 H, ArOH, exchangeable
by D2O); 9.57 (s, 1 H, ArOH, exchangeable by D2O); 9.62 (s,
1 H, ArOH, exchangeable by D2O); 12.10 (s, 1 H, NH, ex-
changeable by D2O).
methoxyphenyl)imidazole 5 a (0.534 mmol (280 mg)).Purifica-
tion was performed by column chromatography on silica gel
with acetone and subsequent re-crystallization from acetone/
CH2Cl2 (5 + 1). Yield: 0.373 mmol (180 mg), 70 %; colorless
powder, mp.: 272–275 °C. IR (KBr): ν = 3600–2600 s, br (OH);
2959 m; 2932 m; 2867 w; 1650 s; 1609 s; 1572 m; 1532 s; 1441
s; 1283 s; 1225 m; 1060 w; 900 m; 856 m; 812 m. MS (EI,
C21H16N2O3 (344.37): calc.: C 73.24 H 4.68 N 8.13, found: C
73.28 H 4.90 N 8.07.
çs
400 °C): m/z (%) = 480 (84) [M]+ ; 446 (10) [M-Cl]+; 410 (7) [M-
4,5-Bis(4-hydroxyphenyl)-2-(2,6-dichloro-3-hydroxyphenyl)-
imidazole (2)
2 Cl]+; 375 (1) [M-3 Cl]+; 265 (11); 231 (31); 139 (36). 1H-NMR
([D6]-DMSO):δ = 6.67 (dd, 3J = 8.5 Hz, 4J = 2.2 Hz, 2 H, ArH-5);
6.74 (d, 4J = 2.2 Hz, 1 H, ArH-3); 6.85 (d, 4J = 2.2 Hz, 1 H, ArH-
3); 6.95 (s, 2 H, ArЈH); 6.97 (d, 3J = 8.5 Hz, 1 H, ArH-6); 7.13 (d,
3J = 8.5 Hz, 1 H, ArH-6); 9.76 (s, br, 1 H, ArOH, exchangeable
by D2O); 9.97 (s, br, 1 H, ArOH, exchangeable by D2O); 10.64
(s, br, 1 H, ArOH, exchangeable by D2O); 12.34 (s, 1 H, NH, ex-
changeable by D2O).
From 4,5-bis(4-methoxyphenyl)-2-(2,6-dichloro-3-methoxy-
phenyl)imidazole 2 a (0.659 mmol (300 mg)).Yield:0.496 mmol
(205 mg), 75 %;colorless powder, mp.:198–202 °C.IR (KBr): ν
3650–2300 s, br (OH);3620 w;1617 s;1568 m;1521 s;1440 s;
1394 m; 1243 s; 1168 m; 1102 m; 835 s; 786 m. 1H-NMR ([D6]-
DMSO): δ = 6.68 (AAЈBBЈ, 3J = 8.5 Hz, 2 H, ArH-3, ArH-5); 6.78
(AAЈBBЈ, 3J = 8.5 Hz, 2 H, ArH-3, ArH-5); 7.09 (d, 3J = 8.8 Hz,
C21H12Cl4N2O3 (482.15): calc.: C 52.31 H 2.51 N 5.81, found: C
52.11 H 2.21 N 5.56.
3
1 H, ArЈH-4); 7.24 (AAЈBBЈ, J = 8.5 Hz, 2 H, ArH-2, ArH-6);
7.31–7.38 (m, 3 H, ArH-2, ArH-6, ArЈH-5); 9.38 (br, 3 H, ArOH,
exchangeable by D2O); 12.30 (s, 1 H, NH, exchangeable by
D2O).
Biological methods
Biochemicals, chemicals and materials
C21H14Cl2N2O3 (413.26): calc.: C 61.03 H 3.41 N 6.78, found:
C 61.06 H 3.62 N 6.86.
Dextran, 17β-estradiol, L-glutamine (L-glutamine solution:
29.2 mg/ml PBS) and Minimum Essential Medium Eagle
(EMEM) were purchased from Sigma (Munich, Germany);Dul-
becco’s Modified Eagle Medium without phenol red (DMEM)
from Gibco (Eggenstein, Germany); Bovine calf serum (BCS)
from Bio whittaker (Verviers, Belgium); N-Hexamethylpara-
rosaniline (crystal violet) and gentamicin sulfate from Fluka
(Deisenhofen, Germany); Glutardialdehyde (25 %) from Merck
(Darmstadt, Germany); Trypsin (0.05 %) in ethylenediamine-
tetraacetic acid (0.02 %) (trypsin/EDTA) from Boehringer
(Mannheim, Germany); Penicillin-streptomycin gold standard
(10000 IE penicillin/mL, 10 mg streptomycin/mL) and geneticin
disulfate (geneticin solution: 35.71 mg/mL PBS) from ICN Bio-
medicals GmbH (Eschwege, Germany); Norit A (charcoal)
from Serva (Heidelberg, Germany); Cell culture lysis reagent
(5×) (diluted 1:5 with purified water before use) and the luci-
ferase assay reagent from Promega (Heidelberg, Germany);
Optiphase HiSafe3 liquid szintillator from Wallac (Turku, Fin-
land); NET-317-Estradiol[2,4,6,7-3H(N)] (17β-[3H]estradiol)
from Du Pont NEN (Boston, Maryland);CDCl3, and [D6]-DMSO
from Aldrich (Steinheim, Germany);Phosphate buffered saline
(PBS) was prepared by dissolving 8.0 g NaCl, 0.2 g KCl, 1.44 g
Na2HPO4 × 2 H2O and 0.2 g KH2PO4 (all purchased from Merck
or Fluka ) in 1000 mL of purified water. TRIS-buffer (pH = 7.5)
was prepared by solving 1.211 g trishydroxymethylaminometh-
an, 0.37224 g Titriplex III and 0.19503 g sodium azide (all from
Merck or Fluka) in 1 L of purified water. Deionized water – pro-
duced by means of a Millipore Milli-QWater System, resistance
> 18 MΩ. T-75 flasks, reaction tubes and 96-well plates were
purchased from Renner GmbH (Dannstadt, Germany).
4,5-Bis(4-hydroxyphenyl)-2-(2,6-dichloro-4-hydroxyphenyl)-
imidazole (3)
From 4,5-bis(4-methoxyphenyl)-2-(2,6-dichloro-4-methoxy-
phenyl)imidazole 3 a (0.527 mmol (240 mg)).Yield:0.484 mmol
(200 mg), 92 %; colorless powder, mp.: 264–266 °C. IR (KBr):
ν= 1650–2500 s, br (OH); 3182 br; 1640 m; 1610 m; 1575 s;
1526 m;1497 s;1455 s;1254 s;1058 m;922 m;834 m.1H-NMR
([D6]-DMSO):δ = 6.67 (AAЈBBЈ, 3J = 8.4 Hz, 2 H, ArH-3, ArH-5);
6.77 (AAЈBBЈ, 3J = 8.3 Hz, 2 H, ArH-3, ArH-5); 6.94 (s, 2 H,
ArЈH); 7.24 (AAЈBBЈ, 3J = 8.3 Hz, 2 H, ArH-2, ArH-6); 7.32
(AAЈBBЈ, 3J = 8.4 Hz, 2 H, ArH-2, ArH-6); 9.60 (br, 3 H, ArOH,
exchangeable by D2O); 12.24 (s, 1 H, NH, exchangeable by
D2O).
C21H14Cl2N2O3 (413.26): calc.: C 61.03 H 3.41 N 6.78, found:
C 60.99 H 3.47 N 6.89.
4,5-Bis(2-fluoro-4-hydroxyphenyl)-2-(2,6-dichloro-4-hy-
droxyphenyl)imidazole (4)
From
4,5-bis(2-fluoro-4-methoxyphenyl)-2-(2,6-dichloro-4-
methoxyphenyl)imidazole 4 a: (0.187 mmol (92 mg)). Purifica-
tion was performed by column chromatography on silica gel
with diethyl ether and acetone (9 + 1) and subsequent re-crys-
tallization from methanol/acetone (1 + 5). Yield: 0.089 mmol
(40 mg), 48 %; colorless powder, mp.: 211–215 °C. IR (KBr): ν
= 3650–2400 s, br (OH); 2933 w; 2678 w; 2585 w; 1628 s; 1600
s; 1521 w; 1457 s; 1306 m; 1245 m; 1155 m; 1114 m; 1063 m;
964 m; 849 m; 812 m. MS (EI, 300 °C): m/z (%) = 448 (100)
çs
[M]+ ; 233 (20); 123 (12); 91 (78). 1H-NMR ([D6]-DMSO): δ =
Estrogen receptor binding assay
6.45 (dd, 3J(H, F) = 12.2 Hz, 4J = 2.1 Hz, 1 H, ArH-3);6.54–6.63
(m, 3 H, ArH-3, 2 × ArH-5);6.96 (s, 2 H, ArЈH);7.07 (dd, 3J = 8.7
Hz, 4J(H, F) = 8.7 Hz, 1 H, ArH-6);7.35 (dd, 3J = 8.7 Hz, 4J(H, F)
= 8.7 Hz, 1 H, ArH-6); 9.76 (s, 1 H, ArOH, exchangeable by
D2O); 9.97 (s, 1 H, ArOH, exchangeable by D2O); 10.64 (s, 1 H,
ArOH, exchangeable by D2O);12.39 (s, 1 H, NH, exchangeable
by D2O).
The applied method was described already by Hartmann et al.
[32] and used with some modifications.The relative binding af-
finity (RBA) of the test compounds to the estrogen receptor was
determined by the displacement of 17β-[3H]estradiol from its
binding site. For this purpose the test compounds were dis-
solved in ethanol and diluted with TRIS-buffer to 6–8 appropri-
ate concentrations (300 µL).They were incubated shaking with
calf uterine cytosol (100 µL) and 17β-[3H]estradiol (0.723 pmol
in TRIS-buffer (100 µL); activity: 2249.4 Bq/tube) at 4 °C over
C21H12Cl2F2N2O3 (449.24): calc.: C 56.15 H 2.69 N 6.24, found:
C 56.18 H 2.47 N 6.05.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

464 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 456–465
night. To stop the reaction 500 µL of a dextran-charcoal-sus-
pension in TRIS-buffer were added to each tube. After shaking
for 90 min at 4 °C and centrifugation 500 µL HiSafe3 was mixed
with 100 µL supernatant of each sample and the reactivity was
determined by liquid scintillation spectroscopy. The same pro-
cedure was used to quantify the binding of 17β-[3H]estradiol
(0.723 pmol – control).Non-specific binding was calculated us-
ing 2 nmol of 17β-estradiol as the competing ligand.On a semi-
log plot the percentage of maximum bound labelled steroid cor-
rected by the non-specifically bound 17β-[3H]estradiol vs. con-
centration of the competitor (log-axis) is plotted. At least six
concentrations of each compound were chosen to estimate its
binding affinity.From this plot those molar concentrations of un-
labeled estradiol and of the competitors, which reduce the bind-
ing of the radioligand by 50 %, were determined
remove the adherent dye. After addition of 180 µL of ethanol
(70 % (v/v)), plates were gently shaken for 4 h. Optical density
of each well was measured in a microplate autoreader at
590 nm (Fash scan, Jena Analytik, Jena, Germany).
The estrogenic activity was expressed as % activation of a
109 M E2 control (100 %).
The anti-estrogenic activity was determined by incubation of
the MCF-7-2a cells with the test compounds in concentrations
from 10–5–10–10 M along with a constant concentration of E2
(10–9 M). IC50 is the concentration of the compound which is
necessary to reduce the effect of E2 by 50 %.
c[3H]-Estradiol at 50 % inhibition
RBA =
· 100 %
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Halide Substituents and Estrogen Receptor Interaction 465
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International Union of Pure and
Applied Chemistry (IUPAC)
P. Heinrich Stahl / Camille G.Wermuth (Eds.)
Handbook of Pharmaceutical Salts
An essential step
in the preclinical phase
of drug development
Properties, Selection, and Use
2002. 388 pages. Hardcover. E 149.00* / sFr 220.00 / £ 85.00.
ISBN 3-906390-26-8.
*The e-Price is valid only for Germany.
O–
Cl
Na+
O
NH
Cl
Contents:
The majority of medicinal chemists in pharmaceutical industry whose primary focus is the
design and synthesis of novel compounds as future drug entities are organic chemists for whom
salt formation is often a marginal activity restricted to the short-term objective of obtaining
crystalline material. Because a comprehensive resource that addresses the preparation, selection,
and use of pharmaceutically active salts has not been available, researchers may forego the
opportunities for increased efficacy and improved drug delivery provided by selection of an
optimal salt.To fill this gap in the pharmaceutical bibliography, we have gathered an international
team of seventeen authors from academia and pharmaceutical industry who, in the contributions
to this volume, present the necessary theoretical foundations as well as a wealth of detailed
practical experience in the choice of pharmaceutically active salts.
The Physicochemical Background: Fundamentals of Ionic
Equilibria • Solubility and Dissolution of Weak Acids, Bases,
and Salts • Evaluation of Solid-State Properties of Salts •
Pharmaceutical Aspects of the Drug Salt Form • Biological
Effects of the Drug Salt Form • Salt-Selection Strategies • A
Procedure For Salt Selection and Optimization • Large-Scale
Aspects of Salt Formation: Processing of Intermediates and
Final Products • Patent Aspects of Drug-Salt Formation •
Regulatory Requirements for Drug Salts in the European
Union, Japan, and the United States • Selected Procedures
for the Preparation of Pharmaceutically Acceptable Salts of
Acids and Bases • Monographs on Acids and Bases
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