Pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs): A new class of agents with high apoptotic activity in chronic myelogenous leukemia K562 cells and in cells from patients at onset and who were imatinib-resistant

Article abstract of DOI:10.1021/jm0602716

Pyrrolo[1,2-b][1,2,5]benzothiadiazepine 5,5-dioxides (PBTDs) induced apoptosis in human BCR-ABL-expressing leukemia cells. The apoptotic activity was also observed in primary leukemic blasts, obtained from chronic myelogenous leukemia (CML) patients at on

Full text of DOI:10.1021/jm0602716

5840
J. Med. Chem. 2006, 49, 5840-5844
Brief Articles
Pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs): A New Class of Agents with High Apoptotic
Activity in Chronic Myelogenous Leukemia K562 Cells and in Cells from Patients at Onset and
Who Were Imatinib-Resistant
Romano Silvestri,*,† Gabriella Marfe`,‡ Marino Artico,† Giuseppe La Regina,† Antonio Lavecchia,§ Ettore Novellino,§
Manuela Morgante,# Carla Di Stefano,‡ Gianfranco Catalano,| Giuseppe Filomeni, Elisabetta Abruzzese,| Maria Rosa Ciriolo,
Matteo Antonio Russo,# Sergio Amadori,| Roberto Cirilli,∞ Francesco La Torre,∞ and Paola Sinibaldi Salimei‡
Istituto PasteursFondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, UniVersita` “La Sapienza”, Piazzale Aldo Moro 5, I-00185
Roma, Italy, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, UniVersita` “Tor Vergata”, Via Montpellier 1, I-00133 Roma, Italy,
Dipartimento di Chimica Farmaceutica e Tossicologica, UniVersita` di Napoli “Federico II”, Via Domenico Montesano 49, I-80131 Napoli,
Italy, Dipartimento di Medicina Sperimentale e Patologia, UniVersita` “La Sapienza”, Viale Regina Elena 324, I-00161 Roma, Italy, Cattedra
di Ematologia, UniVersita` “Tor Vergata”, Ospedale “Sant’Eugenio”, Piazzale dell’Umanesimo 10, I-00144 Roma, Italy, Dipartimento di
Biologia, UniVersita` “Tor Vergata”, Via della Ricerca Scientifica, I-00133 Roma, Italy, and Dipartimento del Farmaco, Istituto Superiore di
Sanita`, Viale Regina Elena 299, I-00161 Roma, Italy
ReceiVed March 10, 2006
Pyrrolo[1,2-b][1,2,5]benzothiadiazepine 5,5-dioxides (PBTDs) induced apoptosis in human BCR-ABL-
expressing leukemia cells. The apoptotic activity was also observed in primary leukemic blasts, obtained
from chronic myelogenous leukemia (CML) patients at onset or from patients in blast crisis and who were
imatinib-resistant. Compounds 5 and 14 induced apoptosis before BCR-ABL protein expression and tyrosin
phosphorylation were affected and activated different caspases in the apoptotic pathway. PBTDs are a new
class of valid candidates for the treatment of CML.
Chart 1
Chronic myelogenous leukemia (CML) is a clonal disease
of hemopoietic progenitor cells.1 CML is characterized by the
expression of the BCR-ABL fusion gene, which is derived from
the fusion of the cellular breakpoint cluster region (BCR) gene
and the Abelson murine leukemia oncogene (ABL).2 BCR-ABL-
transformed cells activate multiple signal transduction pathways
responsible for increasing proliferation. The BCR-ABL protein
also activates downstream survival pathways3 that collectively
provide BCR-ABL cells with a survival advantage over the
normal cells, thereby contributing to the leukemic phenotype4
and conferring higher resistance against conventional cytotoxic
drugs.5
Imatinib (IM, imatinib mesilate, STI-571, Gleevec) (1) is now
the first-choice drug for all newly diagnosed CML patients6
(Chart 1). IM is a potent inhibitor in all of the ABL tyrosine
kinases, including BCR-ABL. There is a lot of evidence that
the oncogene BCR-ABL would protect growth-factor-dependent
hemopoietic cells from apoptosis. According to this role,
inhibition of BCR-ABL expression by IM showed restoration
of susceptibility to apoptosis and enhancement of cell death.7
Exposure to IM alone induced apoptosis in HL-60/BCR-ABL
and K562 cells.8 Despite its initial efficacy, IM selects drug
resistance due to the emergence of mutation at the kinase domain
and overexpression of BCR-ABL.9 To overcome resistance,
some strategies have been adopted, such as dose escalation and
synergistic combination with conventional drugs (cytarabine,
homoharringtonine, IFN), alternative ABL inhibitors, or BCR-
ABL protein down-regulating agents.7
* To whom correspondence should be addressed. Phone: +39 06 4991
3800. Fax: +39 06 491 491. E-mail: romano.silvestri@uniroma1.it.
† Dipartimento di Studi Farmaceutici, Universita` “La Sapienza”.
‡ Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Uni-
versita` “Tor Vergata”.
The antitumor activity of pyrrolo[2,1-c][1,4]benzodiazepines
(PBDs) related to anthramycin (2) was extensively studied by
our research group, as it is well documented in Thurston’s
excellent review.10 A correlation between antitumor activity and
apoptosis was well demonstrated. It was hypothesized that
anthramycin, like other DNA-alkylating agents, would induce
apoptosis through a mythocondrial pathway.11 In addition, in
§ Universita` di Napoli “Federico II”.
# Dipartimento di Medicina Sperimentale e Patologia, Universita` “La
Sapienza”.
| Cattedra di Ematologia, Universita` “Tor Vergata”.
Dipartimento di Biologia, Universita` “Tor Vergata”.
∞ Dipartimento del Farmaco, Istituto Superiore di Sanita`.
10.1021/jm0602716 CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/09/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5841
Table 1. Apoptotic Activity of Compounds 4-18 and Reference
Compound 1 in K562 Cells at 10 µMa
Table 3. Apoptotic Activity of Compounds 5 and 14 at 10 µM in Cells
from CML Patients in Blast Crisis and Who Were Imatinib-Resistant
% of apoptosis ((SD)b
% of apoptosis
compd
8 h
16 h
24 h
48 h
5
14
4
5
6
7
8
9
45.0 ( 2.0
45.6 ( 1.5
44.3 ( 1.5
45.3 ( 2.1
43.7 ( 1.7
45.3 ( 1.1
45.0 ( 2.6
43.0 ( 2.6
44.6 ( 1.5
47.0 ( 2.0
34.0 ( 1.7
29.3 ( 0.6
4.8 ( 0.6
66.6 ( 1.5
67.6 ( 1.5
67.0 ( 2.0
67.6 ( 1.5
66.0 ( 1.0
68.0 ( 1.0
67.6 ( 2.1
65.5 ( 2.1
66.3 ( 1.5
69.0 ( 1.0
61.3 ( 0.6
53.0 ( 1.0
5.7 ( 1.5
80.0 ( 1.0
79.6 ( 1.5
79.7 ( 1.5
81.0 ( 1.0
77.0 ( 1.0
79.5 ( 2.5
74.3 ( 2.5
78.0 ( 1.0
79.0 ( 2.0
80.0 ( 0.0
76.3 ( 1.1
72.0 ( 2.6
6.8 ( 1.5
89.6 ( 0.6
91.7 ( 0.6
91.7 ( 0.6
92.0 ( 1.0
87.6 ( 1.1
92.3 ( 2.1
87.6 ( 1.5
90.0 ( 3.6
90.7 ( 1.7
92.6 ( 3.0
89.7 ( 2.1
88.3 ( 0.6
6.2 ( 2.1
patient
sex
age
sourcea
24 h
48 h
24 h
48 h
13
14
M
F
38
70
PB
PB
60
55
80
78
40
50
60
70
a PB: peripheral blood cells.
Scheme 1a
10
11
12
13
14
15
16
17
18
1c
4.5 ( 0.6
5.5 ( 1.5
6.4 ( 1.5
6.1 ( 2.1
5.3 ( 1.5
66.7 ( 1.5
4.4 ( 0.6
19.3 ( 1.5
70.0 ( 1.0
5.3 ( 1.5
19.6 ( 0.6
71.0 ( 1.5
6.4 ( 1.5
23.0 ( 1.7
71.2 ( 2.1
6.1 ( 1.2
controld
a Compounds 4-18 were tested in parallel with the reference compound
1 as described in Supporting Information. b Arithmetic mean ( standard
deviation (SD) for triplicate experiments. c Imatinib, Gleevec. d Untreated
K562 cells.
Table 2. Apoptotic Activity of Compounds 5 and 14 in Cells from
CML Patients at Onset at 10 µM
% of apoptosis
5
14
patient
sex
age (years)
sourcea
24 h
48 h
24 h
48 h
1
2
3
4
5
6
7
8
9
M
M
F
M
F
F
F
M
M
F
45
60
73
83
46
27
45
35
66
38
65
27
PB
BM
PB
BM
PB
PB
PB
PB
PB
PB
PB
PB
77
70
66
50
60
60
64
65
60
70
55
55
85
85
82
75
80
80
80
85
80
80
78
78
64
50
65
50
50
50
60
60
60
50
52
52
70
70
79
70
70
70
80
80
80
70
71
73
a Reagents and reaction conditions: (a) ethyl glyoxylate dimethoxyacetal,
PTSA, absolute EtOH, reflux, overnight; (b) aroyl chloride, NaHCO3,
1-bromo-3-chloropropane, reflux, overnight; (c) ethyl 3,3-diethoxy-propi-
onate, CH3COOH‚H2O, overnight; (d) aroyl chloride, (i-Bu)3N, dry dioxane,
reflux, 48 h; (e) LiOH, THF‚H2O, room temp, 24 h; (f) LiAlH4, AlCl3,
anhydrous THF, room temp, 3 h.
Chemistry
10
11
12
F
F
Aroylation of the ester 14 with aroyl chlorides in boiling
1-bromo-3-chloropropane in the presence of sodium hydrogen
carbonate afforded the corresponding amides 4-8 (Scheme 1).
The starting ester 14 was prepared by treatment of 1914 with
ethyl 2,2-diethoxyacetate in the presence of 4-toluenesulfonic
acid (PTSA) as a catalyst in boiling absolute ethanol via a
Pictet-Spengler type reaction.15 Derivatives 9-13 were pre-
pared by aroylation of 15 in boiling dioxane in the presence of
triisobutylamine. The starting acetate 15 was obtained by
reaction of 19 with ethyl 3,3-diethoxypropionate in aqueous
glacial acetic acid at 100 °C. Lithium hydroxide hydrolysis of
the esters 14 and 15 afforded the corresponding acids 16 and
17. Lithium aluminum hydride-aluminum chloride reduction
of acid 14 gave alcohol 18. The racemic mixtures 5 and 14
were separated using analytical and semipreparative enantiose-
lective HPLC to give enantiomers 20 and 21, and 22 and 23,
respectively, with >98.0% ee and in 80-90% yield.
a PB: peripheral blood cells. BM: bone marrow cells.
the past few years the activity of some PBDs (i.e., 3)12 in
leukemia cell lines, such as K562 and Jurkat cells, was reported.
Our extensive studies on anti-AIDS agents led to the
discovery of pyrrolo[1,2-b][1,2,5]benzothiadiazepine 5,5-
dioxides (PBTDs) as potent non-nucleoside inhibitors of the
HIV-1 reverse transcriptase.13 Given the high structural similar-
ity between PBTDs and PBDs, we selected two PBTDs (5 and
14) for a preliminary screening aimed to discover new proapo-
ptotic and antileukemia agents. In fact, to our knowledge the
anti-CML properties of PBTDs have not been investigated until
now.
In our first experiments, PBTDs 5 and 14 induced apoptosis
in K562 cells (Table 1). In addition, they efficiently induced
cell death in BCR-ABL-positive leukemia cells taken from
patients who were at onset or were IM-resistant (Tables 2 and
3). These results prompted us to conduct apoptotic mechanism
studies on PBTDs 5 and 14. At the same time we planned the
synthesis of new PBTD derivatives to investigate the effect of
(i) substituents at the 10-aroyl group, (ii) a 1-naphthoyl group
at position 10, and (iii) the introduction of a methylene spacer
group at position 11. Our findings indicate that the PBTDs are
a new class of potential agents for the treatment of CML.
Apoptotic Activity in K562 Cells
The characteristic morphological features of apoptosis, such
as cytoplasmic vacuolization, patterns of chromatin condensation
and micronuclei, and negligible necrosis, were observed upon
treatment of the cells with tested compounds. The chromatin
appeared clumped and was observed at the nuclear periphery.
The samples showed micronuclei or the nuclear body surrounded
by a double membrane and cytoplasmic vacuolization. Large
plasmalemmal blebbing was visible in the cells incubated with

5842 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19
Brief Articles
expression levels. Compound 14 seemed to activate the direct
extrinsic caspase-8 mediated pathway, resulting in faster pro-
teolysis induction.
In contrast, 5 seemed to induce apoptosis through the
mitochondrial intrinsic pathway with a relatively delayed
proteolytic effect (Figure 3S in Supporting Information).
Compounds 5 and 14 Induced Apoptosis by Bypassing
the Apoptotic Suppressor BCR-ABL
Western blot analyses of BCR-ABL and tyrosine phospho-
rylated proteins at different intervals showed that some down-
regulation of the BCR-ABL immunoreactive band was detected
only after 16 h upon treatment with 14 and after 24 h upon
treatment with 5. One point worth noting was that at regular
intervals we noticed a slight diminution of ABL and actin
proteins. At the same time, no significant early change in the
pattern of tyrosine phosphorylated proteins was detected (at least
not until the same BCR-ABL protein level started to fade). This
confirmed that no inhibition of a putative BCR-ABL-mediated
major phosphorylative process was induced within the cell.
These observations suggested that down-regulation of BCR-
ABL is not involved in the early events associated with PTDB-
induced apoptosis. Accordingly, both 5 and 14 induced apoptosis
before the levels of BCR-ABL protein expression and its tyrosin
phosphorylation were affected. Both actin protein degradation
and whole-cell phosphorylation pattern changes (caused by
widespread proteolysis) were dependent on the apoptotic process
rather than the inactivation of BCR-ABL kinase (Figure 4S in
Supporting Information).
In conclusion, our preliminary results demonstrated that
PBTDs 4-15 induced apoptosis in human BCR-ABL-express-
ing leukemia cells. The apoptotic activity was also observed in
primary leukemia blasts, obtained from CML patients at onset
or from patients in blast crisis and who were IM-resistant.
Apoptotic mechanism studies conducted on PBTDs 5 and 14
showed that they activated the caspase activity through two
different pathways. These compounds also induced apoptosis
before the BCR-ABL protein expression and tyrosine phospho-
rylation levels were affected. These findings suggest that PBTDs
are endowed with powerful growth inhibition on leukemia cells.
Figure 1.
5 (Figure 1, left (×18 000)), whereas such morphological
changes did not appear in the control cells (Figure 1, right).
The apoptotic activity of 5 and 14 in K562 cells was time-
dependent at 5, 10, and 15 µM (Figure 1S in Supporting
Information). At 10 µM, 4-15 showed high apoptotic activity
at every time point. A preliminary structure-activity relationship
(SAR) inspection indicated that the apoptotic activity of 4-13
was weakly affected by the aroyl substitutents at position 10 of
the PBTD nucleus or by the methylene linker at position 11
(compare carboxylates 4-8 and acetates 9-13). Compounds
14 and 15 deprived of the 10-aroyl portion also showed potent
apoptotic activity with greater difference after 8 h (compare
4-13 with 14 and 15). Replacement of the ester 14 with the
alcohol 18 caused a neat decrease of apoptotic activity. Free
acids 16 and 17 did not show the morphological features of
apoptosis, showing an apoptotic activity that was comparable
to that of control cells. We hypothesized that these compounds
would not be able to penetrate the K562 cell membrane and to
activate the cellular mechanisms. To evaluate the influence of
the chiral center at position 11 of the PBTD ring, the racemates
5 and 14 were separated by chiral HPLC. Both (+)-(S)-
enantiomers 21 and 23 and the corresponding (-)-(R)-
counterparts 22 and 24 showed slight differences of activity in
the range 3-5% (Table 1S in Supporting Information).
Apoptotic Activity in Bone Marrow and Peripheral Blood
Cells from CML Patients
We harvested bone marrow and peripheral blood cells from
12 consecutive CML patients at onset. We also harvested
peripheral blood cells from 2 patients in blast crisis evolved
under IM treatment. Cell samples were cultured in vitro, treated
with 5 or 14 for 24 and 48 h, and then subjected to DNA
fragmentation assay. Tested PBTDs induced apoptosis in all of
the samples, including those from the two patients in blast crisis
and who were IM-resistant (Tables 2 and 3). The typical
appareance of endonucleosome DNA after treatment with 5, 6,
or 14 in patients cases 1, 11, and 14 is shown in Figure 2S in
Supporting Information.
Besides the high activity displayed by some PBTDs discov-
ered in the present preliminary work, we are aware that further
results are obtainable to evaluate the chemotherapeutic potential
of this novel class of anti-CML agents. For this reason, the
synthesis of new PBTD derivatives is now ongoing in our
laboratories as part of more in-depth structure-activity relation-
ship (SAR) studies guided by molecular modeling.
Experimental Section
Chemistry. Melting points (mp) were determined on a Bu¨chi
510 apparatus and are uncorrected. Infrared spectra (IR) were run
on a SpectrumOne FT spectrophotometer. Band position and
absorption ranges are given in cm-1. Proton nuclear magnetic
resonance (1H NMR) spectra were recorded on Bruker 200 and
400 MHz FT spectrometers in the indicated solvent. Chemical shifts
are expressed in δ units (ppm) from tetramethylsilane. Column
chromatography was performed on columns packed with alumina
from Merck (70-230 mesh) or with silica gel from Merck (70-
230 mesh). Aluminum oxide TLC cards from Fluka (aluminum
oxide precoated aluminum cards with fluorescent indicator at 254
nm) and silica gel TLC cards from Fluka (silica gel precoated
aluminum cards with fluorescent indicator at 254 nm) were used
for thin-layer chromatography (TLC). Elemental analysis results
were found within (0.4% of the theoretical values.
Caspase Activation Activity in K562 Cells
We analyzed the activated apoptotic pathways by performing
Western blot analyses of caspase-8 and -9 proforms. Upon drug
treatment caspase-8 and -9 became activated in most apoptotic
models in receptor-activated caspase-8 and -3 pathways or in
cytochrome c release-dependent caspase-9 and -3 pathways.
In both pathways, caspase-3 activation is the final event of
the caspase cascade. After 24 h, 14 significantly reduced the
procaspase-8 immunoreactive band without changing the pro-
caspase-9 intensity. On the other hand, lysates from 5-treated
K562 cells showed no evidence of alteration in procaspase-8
concentration and paralleled a dramatic decrease of procaspase-9

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 19 5843
References
General Procedure for the Prepapartion of Derivatives 4-8.
Example. Ethyl (()-10,11-Dihydro-10-benzoylpyrrolo[1,2-b]-
[1,2,5]benzothiadiazepine-11-carboxylate 5,5-Dioxide (4). A
mixture of 14 (1.00 g, 0.0033 mol), benzoyl chloride (0.48 g, 0.0034
mol), NaHCO3 (0.31 g, 0.0037 mol), and 1-bromo-3-chloropropane
(50 mL) was refluxed overnight. After cooling, the mixture was
filtered and the solvent evaporated. The residue was purified by
column chromatography (silica gel, chloroform). Yield 40%, mp
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1
172-180 °C (ethanol). H NMR (DMSO-d6): δ 1.30 (t, J ) 7.0
Hz, 3H), 4.28 (m, 1H), 4.44 (m, 1H), 6.16 (m, 1H), 6.28 (br, 1H),
6.34 (m, 1H), 7.06 (d, J ) 6.9 Hz, 1H), 7.25-7.51 (m, 8H), 7.91
ppm (dd, J ) 1.5 and 7.4 Hz, 1H). IR (neat): ν 1185, 1650, 1745
cm-1. Anal. (C21H18N2O5S (410.44)), C, H, N, S.
General Procedure for the Prepapartion of Derivatives 9-13.
Ethyl (()-10,11-Dihydro-10-benzoylpyrrolo[1,2-b][1,2,5]benzo-
thiadiazepine-11-acetate 5,5-Dioxide (9). A mixture of 15 (1.00
g, 0.0031 mol), benzoyl chloride (0.56 g, 0.0040 mol), triisobuty-
lamine (0.74, 0.0040 mol), and dry dioxane (50 mL) was refluxed
for 48 h. After concentration the mixture was extracted with ethyl
acetate, washed with 1 N HCl and then with brine, and dried.
Removal of the solvent furnished a residue that was purified by
column choromatography (silica gel, dichloromethane). Yield 35%,
1
mp 178-181 °C (ethanol). H NMR (DMSO-d6): δ 1.19 (t, J )
7.1 Hz, 3H), 2.81 (m, 1H), 2.91 (m, 1H), 4.12 (m, 2H), 6.35-6,40
(m, 2H), 6.56 (br, 1H), 7.22-7.58 (m, 9H), 8.00 ppm (m, 1H). IR
(neat): ν 1186, 1315, 1655, 1725 cm-1. Anal. (C22H20N2O5S
(424.47)), C, H, N, S.
Ethyl (()-10,11-Dihydropyrrolo[1,2-b][1,2,5]benzothiadiaz-
epine -11-carboxylate 5,5-Dioxide (14). A mixture of 19 (9.4 g,
0.042 mol), dimethoxyacetal of ethyl glyoxylate (11.10 g, 0.063
mol), and 4-toluenesulfonic acid monohydrate (PTSA) (8.0 g, 0.042
mol) was refluxed in absolute ethanol (20 mL) overnight. PTSA
(3.7 g, 0.021 mol) was added while refluxing for an additional 4 h.
After cooling, the mixture was poured onto crushed ice and
extracted with ethyl acetate, washed with brine, and dried. Evapora-
tion of the solvent furnished the crude product, which was purified
by column chromatography (silica gel, chloroform). Yield 51%,
mp 131-134 °C (toluene/ligroin) (lit.16a mp 121-122 °C).
Ethyl (()-10,11-Dihydropyrrolo[1,2-b][1,2,5]benzothiadiaze-
pine-11-acetate 5,5-Dioxide (15). 15 was prepared as 14 using ethyl
3,3-diethoxypropionate by heating in aqueous acetic acid. Yield
45%, mp 121 °C (from benzene/petroleum ether) (lit.16b mp 118-
119 °C).
(()-10,11-Dihydropyrrolo[1,2-b][1,2,5]benzothiadiazepine-11-
carboxylic Acid 5,5-Dioxide (16). Lithium hydroxide monohydrate
(0.25 g, 0.006 mol) was added to a solution of 14 (0.61 g, 0.002
mol) in THF (20 mL) and water (20 mL). Then the reaction mixture
was stirred at room temperature for 24 h. After dilution with water,
the mixture was acidified with 1 N HCl until pH 2 was reached.
The acid was extracted with ethyl acetate, washed with brine, and
dried. Removal of the solvent gave pure 16. Yield 98%, mp 161-
163 °C (aqueous ethanol) (lit.16c mp 159-162 °C).
(()-10,11-Dihydropyrrolo[1,2-b][1,2,5]benzothiadiazepine-11-
acetic Acid 5,5-Dioxide (17). 17 was prepared as 16 starting from
15. Yield 90%, mp 208-210 °C (ethanol) (lit.16b mp 209 °C).
(()-10,11-Dihydro-11-hydroxymethylpyrrolo[1,2-b][1,2,5]-
benzothiadiazepine 5,5-Dioxide (18). To anhydrous aluminum
chloride (0.71 g, 0.005 mol) in THF (30 mL) at 0 °C was added
lithium aluminum hydride (8.0 mL of 1 M in THF) while stirring
for 15 min. Then a solution of 16 (0.25 g, 0.000 92 mol) in THF
(10 mL) was added and the mixture was stirred at room temperature
for 3 h. After cooling at 0 °C, the reaction mixture was quenched
with 4 N sodium hydroxide and extracted with dichloromethane.
The organic layer was separated, washed with brine, and dried.
Removal of the solvent gave crude 19, which was passed through
a chromatographic column (silica gel, chloroform). Yield 87%, mp
136-138 °C (ethanol) (lit.13 mp 135-136 °C).
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