Selective naphthalene H3 receptor inverse agonists with reduced potential to induce phospholipidosis and their quinoline analogs

Article abstract of DOI:10.1016/j.bmcl.2009.03.100

We reported earlier the refinement of our initial five-point pharmacophore model for the Histamine 3 receptor (H₃R), with a new acceptor feature important for binding and selectivity against the other histamine receptor subtypes 1, 2 and 4. This approach was validated with a new series of H₃R inverse agonists: the naphthalene series. In this Letter, we describe our efforts to overcome the phospholipidosis flag identified with our initial lead compound (1a). During the optimization process, we monitored the potency of our molecules toward the H₃ receptor, their selectivity against H₁R, H₂R and H₄R, as well as some key molecular properties that may influence phospholipidosis. Encouraged by the promising profile of the naphthalene series, we used our deeper understanding of the H₃R pharmacophore model to lead us towards the quinoline series. This series is perceived to have intrinsic advantages with respect to its amphiphilic vector.

Full text of DOI:10.1016/j.bmcl.2009.03.100

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4495–4500
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Selective naphthalene H3 receptor inverse agonists with reduced potential
to induce phospholipidosis and their quinoline analogs
*
Rosa María Rodríguez Sarmiento , Matthias H. Nettekoven, Sven Taylor, Jean-Marc Plancher, Hans Richter,
Olivier Roche
Hoffmann-La Roche Ltd, Pharmaceutical Research Basel, Discovery Chemistry, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
We reported earlier the refinement of our initial five-point pharmacophore model for the Histamine 3
receptor (H3R), with a new acceptor feature important for binding and selectivity against the other his-
tamine receptor subtypes 1, 2 and 4. This approach was validated with a new series of H3R inverse ago-
nists: the naphthalene series. In this Letter, we describe our efforts to overcome the phospholipidosis flag
identified with our initial lead compound (1a). During the optimization process, we monitored the
potency of our molecules toward the H3 receptor, their selectivity against H1R, H2R and H4R, as well as
some key molecular properties that may influence phospholipidosis.
Encouraged by the promising profile of the naphthalene series, we used our deeper understanding of
the H3R pharmacophore model to lead us towards the quinoline series. This series is perceived to have
intrinsic advantages with respect to its amphiphilic vector.
Received 9 February 2009
Revised 18 March 2009
Accepted 21 March 2009
Available online 26 March 2009
Keywords:
Histamine H3 receptor
SAR
Pharmacophore model
Naphthalene
Ó 2009 Published by Elsevier Ltd.
Phospholipidosis
Amphiphilic vector
Amphiphilicity
Quinoline
Histamine elicits physiological responses mediated by four G-
protein coupled receptors (H1R, H2R, H3R and H4R).1 The histamine
H1 and H2 receptors are well established drug targets2 and the H4
receptor is currently undergoing rigorous characterization.3 H3
receptors are expressed predominantly on the presynaptic termini
of CNS neurons, where they function as inhibitory auto- and het-
eroreceptors.4,5 H3 antagonists/inverse agonists can therefore func-
tion to increase the release of various neurotransmitters, including
histamine, acetylcholine, norepinephrine, serotonin and dopa-
mine.6–10 These neurotransmitters are known to play important
roles in vigilance, attention, cognition and energy homeostasis. In
this regard, a remarkable arrays of therapeutic areas in which li-
gands for the H3R may prove useful have been identified.
Initial work in the field focused on analogs of the natural ligand,
histamine. Due to the presence of the imidazole ring, these com-
pounds have numerous liabilities and poor drug-like properties.
Second generation H3R antagonists are devoid of an imidazole-
moiety, but contain one or two basic sp3 nitrogen atoms. Several
compounds of this class are currently being developed and recently
entered into human clinical trials for the potential treatment of a
variety of CNS disorders affecting cognition (e.g., schizophrenia,
attention-deficit hyperactivity disorder and Alzheimer’s dementia)
and sleep (e.g., hypersomnia, narcolepsy).11 H3R inverse agonists
may also be useful in the control of food intake and obesity.12
We identified the naphthalene class of compounds as selective
and potent H3R inverse antagonists from the refinement of our ini-
tial five point pharmacophore model13 by the use of the acceptor
functionality in close vicinity to the largest aromatic feature
(Fig. 1), important for the binding and the selectivity against H1R,
H2R and H4R.14
Searching for potent compounds, we identified the (3-piperi-
din-1-yl-propoxy)-naphthalene derivative 1a14 as a lead structure.
In fact, compound 1a has many favorable properties: high solubil-
ity,15 high permeability,16 and high metabolic stability in rat and
human liver microsomes.17 In terms of safety, this compound did
not have any issues with respect to mutagenicity (genotoxicity
and clastogenicity)18 or phototoxicity in vitro (Fig. 2).
However, the combination of the lipophilic naphthalene core
with a basic nitrogen tail, protonated at physiological pH, would
result in this class of compound being amphiphilic. Cationic
amphiphilic drugs are associated with phospholipidosis19 and in-
deed compound 1a was found to induce phospholipidosis in cul-
tured fibroblasts at test concentrations of 2.5–20
lM in a
concentration dependent manner.20
* Corresponding author. Tel.: +41 61 6886227; fax: +41 61 6886459.
E-mail address: rosa.maria_rodriguez_sarmiento@roche.com (R.M. Rodríguez
Sarmiento).
Phospholipidosis may have adverse physiological consequences
and has already been reported in association with some H3R
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.03.100

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R. M. Rodríguez Sarmiento et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4495–4500
Figure 1. Compound 1a of the naphthalene class fitting the latest pharmacophore model described previously.14
Table 1
O
Exploration of in vitro potency, selectivity, lipophilicity, basicity and in silico DDGAM
for the naphthalene class
N
O
N
O
N
1a
R
N
O
In vitro profile:
hH3 Ki= 7nM 21 , GTPγS: EC50= 200 nM 22
1
b
c
inverse agonist
Compound hH3
R
hH1/hH2/hH4 KOW_Clog pKa DDGAM
inhibitiona (%)
P
(kJ/mol)
Selectivity
Ki
(nM)
H1 / H2 / H4: -14.7 / -0.3 / 0%inhib@3uM
LYSA: >543 ug/mL
PAMPA Peff: 2.6 10-6 cm/s (high permeability)
logD7.4 : 2.47
1a
1b
7
À14.7/0.3/0
À4.5/4.1/0
5.3
5.1
9.86 À10.4
microsomes:
Clearance
36
9.8
À9.7
human 32 uL/min/mg protein
rat 33 uL/min/mg protein
IC50 CYP3A4, 2D6, 1A2, 2C9, 2C19: all >12μM
N
not phototoxic in vitro
1c
1d
1e
1f
571
234
34
—
4.6
4.5
4.3
4
9.9
9.4
9.6
7.8
À10.1
À9.4
À9.5
À10
no liabilities in Ames and MNTtest
Phospholipidosis induction : positive @2.5uM,
N
strongly positive @5 uM and above
N
—
Figure 2. Structure and properties of compound 1a (See above-mentioned refer-
ences for further information.)
ligands.23 The use of the amphiphilicity as a predictor for phospho-
lipidosis liability has already been widely established19,24 and
therefore we decided to closely monitor the amphiphilicity of the
naphthalene class, which was calculated by a computational tool
(CAFCA).25
0.9/À8/0
N
N
2000
—
O
We investigated the influence of the pKa26 on the amphiphilicity
by changing the nature of the nitrogen tail (R group on Table 1).
Unfortunately, the calculated free energy of amphiphilicity
a
b
c
Percentage measured at 10 lM.
Measured pKa.
Calculated free energy of amphiphilicity as measure of phospholipidosis
potential.
(
DDGAM) for the compounds in Table 1 was always below À6 kJ/
mol. We aimed for DDGAM values greater than À6 kJ/mol, as it
has been found that such a threshold ensures a low probability
of being positive on the fibroblast phospholipidosis assay, where-
no influence on the phospholipidosis hazard, as long as the pKa is
larger than 6.3.24 However, such a low pKa value might prevent
the compound from binding as the formation of the Asp3.32 salt
bridge (i.e., the formation of an ionic bond between the protonated
nitrogen of the ligand and the anionic acid Asp3.32) is no longer
secured.
Moreover, as it can be seen in Table 1, the calculated lipophilic-
ity, expressed as KOW_Clog P, is not correlated with the
amphiphilicity.
as DDGAM values below À6 kJ/mol indicate
a high risk of
phospholipidosis.24
It was observed within the series that significant modifications
of the pKa (ca. 2 log units) only marginally improved DDGAM (see
Table 1, cpds 1a vs 1f). When basicity was highly reduced (com-
pound 1f with the 3-morpholinyl-propoxy has a lower pKa) amph-
iphilicity was not further improved and micromolar activity at H3R
was obtained. Compound 1f with a pKa of 7.8 is still cationic at
physiological conditions and therefore amphiphilic. As a conse-
quence, it can be envisaged that the reduction of pKa value has
We carried on with the exploration of the amide side chain,
combined with the two most potent basic tails: the piperidinyl
propoxy and the isopropyl piperidinoxy (R groups in Table 2).

R. M. Rodríguez Sarmiento et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4495–4500
4497
Table 2
Second round on the exploration of in vitro potency and amphiphilicity. In silico DDGAM
O
R1
R
O
Compound
hH3 Ki (nM)
R1
R
KOW_ClogP
pKa
DDGAM (kJ/mol)
À10.4
N
N
N
1a
7
5.28
9.86
N
HO
O
2
3
103
30
3.29
3.24
9.6
À5.9
À6.7
N
9.57
N
4
5
6
7
15
154
62
3.98
2.55
2.33
2.10
9.71
9.6
À9. 07
À6.1
N
N
N
N
N
HO
N
9.4
À6.5
O
O
N
40
9.3
À4.27
N
These two R groups have different conformational restrictions, and
they also introduce slightly different distances from the cationic
charge to the hydrophobic center.
For all the 3-piperidinyl propoxy side chains, amphiphilicity
was highly reduced with the use of more hydrophilic amides (2
and 3 compared to 1a). Unfortunately, the potency was lost when
DDGAM was greater than À6 kJ/mol (compound 2, Table 2). Com-
pound 3 is still sufficiently active and has an acceptable and im-
proved amphiphilicity.
Compound 7 is potent in vitro at the H3R, and selective against
H1R, H2R and H4R. It has many favorable properties such as high
solubility, high permeability and high metabolic stability in rat
and human liver microsomes, and a clean safety profile, like the
initial lead compound 1a. However, in contrast to 1a, 7 has a much
reduced amphiphilicity, and no flag in phospholipidosis (Fig. 3).
Despite this achievement, we decided to investigate other central
cores, to potentially overcome some of the limitations from the
naphthalene scaffold, particularly with regard to phospholipidosis.
The use of an isopropyl piperidinyloxy as the basic nitrogen tail
introduced a slightly different distance from the cationic charge to
the hydrophobic center and marginally reduced the amphiphilic
vector (compounds 5 vs 2; compounds 6 vs 3 in Table 2; compounds
1e vs 1a in Table 1). This effect becomes slightly synergistic when
combined with the introduction of more hydrophilic amides at the
terminal side of the molecule (compound 4 compared with 1a in Ta-
ble 2). Compound 4 has a slightlyshorter nitrogen tail (thus a slightly
reduced amphiphilic vector) but it is still quite lipophilic, even if
much less than 1a, so that the absolute value for DDGAM is signifi-
cantly reduced but still very high. Unfortunately, less potent com-
pounds are obtained when the lipophilicity is further reduced
(from the methyl pyrrole compound 4 to the hydroxyl methyl pipe-
ridinyloxy analog 5 and the 3-methoxy piperidinyl analog 6).
The combination of the morpholine amide with the isopropyl
piperidinyloxy as the nitrogen tail leads to compound 7 with re-
duced lipophilicity and an appropriate DDGAM greater than
À6 kJ/mol, indicating a low risk for phospholipidosis. This com-
pound has an acceptable potency on binding and behaves as an
H3R inverse agonist.
O
N
N
O
O
7
In vitro profile:
hH3 Ki= 40nM 21; GTPγ
22
S: EC50= 17nM
inverse agonist
Selectivity
H1 / H2 / H4: 1.6 / -9.9 / 0%inhib@3uM
LYSA: >558ug/mL
PAMPA Peff: 2.5 (high permeability)
logD7.4 : 0.6
microsomes:
Clearance
human: 3 uL/min/mg protein
rat : 23 uL/min/mg protein
CYP3A4, 2D6, 1A2, 2C9, 2C19: all > 41 uM
not phototoxic in silico
It is obvious that the substituents on the amide side chain in the
naphthalene series play a key role on the lipophilicity, and even
more on the amphiphilicity of the compound (compounds 1a, 2,
3 and also 4, 5, 6, 7).
no liabilities in Ames and MNT tests
Phospholipidosis: No flag for induction
Figure 3. Structure and properties of compound 7.

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R. M. Rodríguez Sarmiento et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4495–4500
O
a
b
O
O
c
O
OH
N+
O
N
N
11
N
N
O
8
9
10
d,e
O
O
HO
R1
N
f
R
(i,j)
R
OEt
OEt
R2
N
N
N
O
O
O
13
12
(intermediate of 17,18 and 20,21,22)
g
(i,j)
N
O
h
Cl
O
OEt
N
OEt
N
O
O
15
14
(intermediate of 16 and 19)
Scheme 1. Reagents and conditions: (a) CH3COOH, H2O2, reflux, 2 h, 81%;(b) benzoyl chloride, AgCN, rt 4 h–refluxing overnight, 81% or alternative Me3SiCN, Me2NCOCl,
CH2Cl2, rt, 87% (c) (i) NaOH, MeOH, 90 °C, 90%; (ii) HCl (25%), H2O; (d) HBr, reflux, 48 h, (alternative microway, 160 °C, 1 h), 95%; (e) H2SO4, EtOH, reflux, 83%; (f) ROH, PPh3, di-
ethyl azodicarboxylate, THF, 60–80%; or two step procedure with (g) Br(CH2)3Cl, K2CO3, butanone, rt 90%; (h) piperidine, K2CO3, 70%; (i) HCl dioxane, reflux; (j) CDI, R1R2NH2,
DMF (60–85%yields).
The H3R pharmacophore model indicated that the modification
of the central core should be possible, and we hypothesized that
quinoline analogs could be of interest. Replacement of the naph-
thalene core by a quinoline should have a positive impact on the
phospholipidosis profile of our compounds by reducing the hydro-
phobicity of the central core, thus reducing the amphiphilic vector.
General synthesis for these new analogs is indicated in Scheme 1.
Initially the conversion of 6-methoxy-quinoline 8 to 2-cyano-6-
methoxyquinoline 10 was performed without isolation of the
intermediate Reissert compound. This involved treating 6-meth-
oxyquinoline with tosyl chloride, potassium cyanide and water in
dichloromethane for one week at room temperature.27 This re-
ported reaction gave in our hands only a 31% yield. Moreover,
the procedure was not suitable for scaling up.
An improved synthesis of the quinoline analogs started from
the commercially available 6-methoxy-quinoline 8, which was
converted to the N-oxide 9 by treatment with hydrogen peroxide
in acetic acid. Modification of the ortho-alkylation procedure by
using benzoyl chloride as activating agent and silver cyanide in
chloroform afforded the desired 6-methoxy-quinoline-2-carboni-
trile 10.28 Later on with the use of TMSCN and diisopropycarba-
moyl chloride compound 10 was obtained in 87% yield.
Hydrolysis of the cyanide to the carboxylic acid 11 was per-
formed by treatment with sodium hydroxide in methanol. The
phenolic function was liberated in refluxing hydrobromic acid in
48 h. This reaction was highly accelerated with microwave irradi-
ation at 160 °C. Esterification under usual conditions to make the
compound easier to manipulate yielded 6-hydroxy-quinoline-2-
carboxylic acid ethyl ester 12 in high yield. The basic side chain
was introduced by the use of Mitsunobu conditions with the cor-
responding alcohol, or in a two step procedure by alkylation with
1-bromo-3-chloropropane using potassium carbonate to the O-
alkylchloride 14, followed by heating with the corresponding
amine to prepare intermediate 15. The amide was formed after
saponification or acidic hydrolysis of the ester to the 6-alkoxy-
quinoline-2-carboxylic acid; in our hands the use of carbonyl
diimidazole as coupling reagent gave high yields of the final
compounds.
The direct comparison between the naphthalene and the quin-
oline series with the same substitution pattern at R and R1 (1a vs
16; 6 vs 17; 7 vs 18) indicates that our initial hypothesis about
the reduction of the lipophilicity for the central core was correct
and accompanied by a reduction of the amphiphilicity, which im-
proved the phospholipidosis profile. Unfortunately, the potency
at the H3R was also reduced for the quinoline series compared to
its naphthalene analog.
The Structure–Activity Relationship (S.A.R.) of the in vitro po-
tency for this new class of compounds is slightly different to the
naphthalene class, and a new series of derivatives were prepared
in order to study and to improve the potency at the H3R, as indi-
cated in Table 3. The replacement of the basic isopropyl piperidi-
nyloxy for a bicyclic analog improved the potency at the H3R
(compound 18 is less potent than 22) and had a similar effect on
the calculated free energy of amphiphilicity DDGAM (kJ/mol).
In conclusion, we presented the delicate balance existing be-
tween amphiphilicity and H3R affinity in these series. Extensive
S.A.R. investigations around the naphthalene core were rewarded
by the identification of compound 7 as a potent H3R inverse ago-
nist, with no phospholipidosis flag and a promising overall profile.
The encouraging profile of the naphthalene series and the use of
our deeper understanding of the pharmacophore model for the
H3R led us towards another series of compounds with interesting
activity as H3R inverse agonists: the quinoline series. This series
showed an improvement in the phospholipidosis profile, as was
predicted from the observation of the general effect of the lipophil-
icity of the central core on the amphiphilicity. However, this series
seems to have a lower potency in direct comparison of analogs
from the naphthalene class.

R. M. Rodríguez Sarmiento et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4495–4500
4499
Table 3
Quinoline series. Lipophilicity and amphiphilicity calculated by DDGAM
O
N
1
R
R
R
O
Compound
hH3 Ki (nM)
R1
R
KOW_Clog P
pKa
DDGAM (kJ/mol)
À9.4
N
N
16
300
4.47
9.7
N
17
18
125
238
1.53
2.18
9.5
9.5
À5.06
À3.2
N
O
O
N
N
N
19
20
119
62
2.84
2.09
9.4
9.5
À5.4
À4.7
N
N
N
N
N
21
22
87
30
2.24
2.5
9.5
À3.21
À3.4
N
H
8.97
O
N
N
H
9. Threlfell, S.; Cragg, S. J.; Kallo, I.; Tur, G. F.; Coen, C. W.; Greenfield, S. A. J.
Neurosci. 2004, 24, 8704.
Acknowledgments
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Pharmacol. Exp. Ther. 2005, 313, 176; (b) Ligneau, X.; Perrin, D.; Landais, L.;
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D.; Brioni, J. D. Br. J. Pharmacol. 2008, 154, 1; (b) Celanire, S.; Wijtmans, M.;
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15. Solubility measurements were performed by a method developed in house
from a 10 mM DMSO stock solution. This method is similar to the classical
thermodynamic shake-flask solubility, with the only difference being that
DMSO is removed before measurement by an additional lyophilization step.
The assay is called lyophilizated solubility assay (LYSA).
16. Measurements were performed according to Kansy, M.; Senner, F.; Gubernator,
K. J. Med. Chem. 1998, 41, 1007.
17. Measurements were performed according to Obach, R. S.; Baxter, J. G.; Liston, T.
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18. Genotoxicity is estimated with the use of the AMES microsuspension assay,
where the read-out parameter is the increase in the number of revertant
colonies (mutation frequency) of treated compared to untreated control in five
different Salmonella typhimurium tester strains.The micronucleus in vitro test
(MNT in vitro test) is used to detect chromosomal damage. The read-out
It is with real pleasure that we wish to thank all our collabora-
tors whose contributions made the work described possible and so
enjoyable, especially Susanne Raab and Christoph Ullmer. Also spe-
cial thanks to Dr. Holger Fischer for his valuable comments.
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parameter is the increase in the number of micronucleated cells (%) of treated
compared to untreated control in cultivated mammalian cells.
150
a total volume of 180
Nonspecific binding was measured in the presence of 10
were sealed and agitated (350 rpm) at room temperature for 2 h. The beads
were then settled by centrifugation (1000 rpm, 10 min) and the plate counted
in a top count using quench correction.
l
g protein/mL). GTPg35S binding was performed in 96-well microplates in
L with 30 g membrane proteins and 0.28 nM GTP 35S.
M cold GTP S. Plates
l
l
c
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cationic amphiphilic drugs (CAD) like amiodarone, clomipramine, perhexilline,
tamoxifen. CAD’s can be described by two fundamental physico-chemical
properties, the basic pKa value reflecting the cationic nature of the molecule
and it’s amphiphilicity that is defined as the distance between the charged
l
c
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phospholipidosis assay. With this approach approximately 80% of the
positive and negative in vitro findings could be classified correctly. For a
more detail description see: Fischer, H.; Kansy, M.; Potthast, M.; Csato, M. In
Proceedings of the 13th European Symposium on Quantitative Structure–Activity
Relationships, Duesseldorf, Germany, August 27–September 1, 2000; Prous
Science: Barcelona, Spain, 2001.
residue and the more remote hydrophobic residues. For
a more detailed
description see: Muster, W.; Breidenbach, A.; Fischer, H.; Kirchner, S.; Mueller,
L.; Paehler, A. Drug Discovery Today 2008, 13, 303.
20. (a) Handrock, K.; Lüllmann-Rauch, R.; Vogt, R. D. Toxicology 1993, 85, 199; (b)
Lüllmann-Rauch, R.; Pods, R.; von Witzendorff, B. Toxicology 1996, 110, 27; (c)
Mason, R. J.; Walker, S. R.; Shields, B. A.; Henson, J. E.; Williams, M. C. Am. Rev.
Respir. Dis. 1985, 131, 786.
21. Saturation binding experiments were performed using HR3-CHO membranes
prepared as described in Takahashi, K; Tokita, S.; Kotani, H. J. Pharmacol. Exp.
Ther. 2003, 307, 213. All compounds were tested at a single concentration in
duplicates. Compounds that showed an inhibition of [3H]-RAMH by more than
50% were tested again to determine IC50 in a serial dilution experiment. Ki’s
were calculated from IC50 based on the Cheng–Prusoff equation: Cheng, Y;
25. A program called CAFCA (CAlculated Free energy of Charged Amphiphiles) was
developed and used for the calculation of the amphiphilic properties of
molecules. The amphiphilicity is the vector sum calculated from the charged
group to each atom/residue within a molecule and weighted with respect to its
hydrophobic/hydrophilic property on the basis of an atom/fragment based
contribution method. The sum of the calculated vectors is calibrated by means
of measured amphiphilicities taking into account the conformational effects of
the individual molecules. Finally, the amphiphilicity of a molecule is expressed
in terms of free energy (DDGAM). For a more detail description, see: Fischer, H.;
Kansy, M.; Bur, D. Chimia 2000, 54, 640.
Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099. For
a more detailed
description see: Gatti, S.; Hertel,C.; Nettekoven, M.; Plancher, J.-M.; Raab, S.;
Roche, O.; Rodriguez-Sarmiento, R.-M. PCT Int. Appl. 2005, WO2005117865.
22. The GTPcS-binding assay was performed using membranes from CHO cells
(Euroscreen, Belgium). After thawing, the membranes were suspended in
20 mM HEPES–NaOH buffer (pH 7.4) containing 1 mM MgCl2, 100 mM NaCl,
26. Measurements were performed according to: Allen, R. I.; Box, K. J.; Comer, J.;
Peake, C.; Tam, K. Y. J. Pharm. Biomed. Anal. 1998, 17, 699.
45
lg/mL Saponin and 10
lM GDP. Membrane suspension and wheat germ
27. Boger, D.; Panek, J. J. Am. Chem. Soc. 1985, 107, 5745.
agglutinin SPA beads (Amersham) were mixed (beads 13 mg/mL; membranes
28. Montanari, F.; Pentimalli, L. Gazz. Chim. Ital. 1953, 83, 273.