Discovery and SAR of cyclic isothioureas as novel NPY Y1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.09.048

A class of novel 2-aminobenzothiazoles have been identified as NPY Y₁ antagonists. Various N-heterocyclic substituted aminophenethyl-2-aminobenzothiazole analogs were synthesized to explore the SAR. Isothiourea analogs and ligands with high potency (K_i 30 nM) have been identified.

Full text of DOI:10.1016/j.bmcl.2009.09.048

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6801–6805
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of cyclic isothioureas as novel NPY Y1 receptor antagonists
a
a
a
a
Zhong-Yue Sun a, , Zhaoning Zhu , Yuanzan Ye , Brian McKittrick , Michael Czarniecki ,
*
William Greenlee a, Deborra Mullins b, Mario Guzzi b
a Department of Medicinal Chemistry, Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
b Department of Neurobiology, Schering Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A class of novel 2-aminobenzothiazoles have been identified as NPY Y1 antagonists. Various N-heterocy-
clic substituted aminophenethyl-2-aminobenzothiazole analogs were synthesized to explore the SAR.
Isothiourea analogs and ligands with high potency (Ki 30 nM) have been identified.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 10 August 2009
Revised 9 September 2009
Accepted 14 September 2009
Available online 25 September 2009
Keywords:
NPY Y1 receptor antagonist
Thioisourea
Thiazoline
Thiazine
Neuropeptide Y (NPY), a 36 amino acid polypeptide, is a member
of a peptide family that includes peptide YY (PYY) and pancreatic
polypeptide (PP).1 NPY is widely distributed in the central and the
peripheral nervous system, and has been implicated in the regula-
tion of food intake, energy metabolism, cardiovascular function,
neuronal excitability and endocrine function.2 There are five NPY
receptors that have been cloned (Y1, Y2, Y4, Y5 and Y6), and more than
one receptor subtype seems to be involved in the regulation of feed-
ing.3 Amongthese, NPYY1 is thoughttobe animportantmajorrecep-
tor, since various NYP Y1 antagonists are found to inhibit food
intake.4 Therefore, identification of NPY Y1 antagonists would have
implications in the treatment of obesity. After the identification of
BIBP3226, an arginine derivative as the first non-peptide NPY Y1
antagonist,5 a number of small molecule NPY Y1 antagonists,
including dihydropyridines,6 indoles,7 benzimidazoles,8 pyrazolo-
pyrimidines,9 aminopyridines,10 benzazepines11 and tetrahydrocar-
bazoles,12 have been described. Here we report another novel
structural series of selective NPY Y1 antagonist.
Isothiourea 1a with a Ki of 248 nM for NPY Y1 binding was iden-
tified through a high throughput screening campaign. Compound
1a displayed an antagonist profile determined by a functional
assay with a Kb of 58.5 nM (Table 3), and was highly selective
against NPY Y2, Y4, and Y5 receptors (Table 3). This molecule has
a 2-aminobenzothiazole on one end and a five-membered cyclic
isothiourea group on the other, bridged with a flexible phenethyl
linker (Fig. 1).
Isothiourea 1a, with its multiple points for solid phase attach-
ment, holds potential for efficient solid phase synthesis. Therefore,
we developed multiple mild solid phase methodologies to explore
a diverse SAR space that varied both ends of the molecules (2 and
3). Therefore, 2-aminobenzothiazole (5) was loaded onto p-nitro-
phenol carbonate Wang resin (4) to form the resin bound carbamate
6. Theacidity of thebenzothiazoleamino group, activatedby the car-
bamate linker, is high enough to undergo Mitsunobu alkylation13
with 2-(p-aminophenyl)ethanol to give the resin bound p-amino-
phenethyl-2-aminobenzothiazole 7. The resin loading level was
0.46 mmol/g based on the recovered p-aminophenethyl-2-amino-
benzothiazole (7a) obtained from resin cleavage using TFA. The resin
boundbenzoylthiourea8, thiourea9 andisothiocyanate 10were ob-
tained by treating 7 with benzoyl isothiocyanate, with Fmoc-isothi-
ocyanate followed by Fmoc de-protection, and with di-2-
pyridylthionocarbonate,14 respectively (see Scheme 1).
We first attempted the replacement of the thiazoline in 1a with
other groups such as amines, amides, ureas or sulfonamides with-
out success (data not shown). We then turned our attention to het-
erocycles structurally related to the cyclic isothiourea in 1a, such
as imidazolines, imidazolones, thiazoles and 4-thiazolones repre-
sented by 11, 12, 13 and 14 in Schemes 2 and 3.
Therefore, resin bound benzoyl thiourea 8, activated by the ben-
zoyl group on thiourea nitrogen, reacted with
a-aminoalcohols
using EDCI as a coupling reagent to form the corresponding ben-
zoyl guanidines,15 which were then cyclized under Mitsonobu
reaction condition to give the corresponding N-benzoyl imidazo-
lines (11) after TFA cleavage and purification (Scheme 2). Similarly,
when
a-aminoesters were used in this scheme, the intermediate
* Corresponding author. Tel.: +1 908 740 3489.
E-mail address: zhong-yue.sun@spcorp.com (Z.-Y. Sun).
benzoyl guanidines readily cyclize after treatment with NaOEt in
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.048

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Z.-Y. Sun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6801–6805
H
N
S
S
N
N
N
H
1a
R5
N
H
N
H
N
n= 0, 1
R4'
R4
S
S
S
NH
Het
N
R3
N
N
H
3
R2
R1
2
Figure 1. SAR exploration on the basis of the lead 1a.
O
S
N
O
N
O
S
NH2
S
NO2
HN
O
O
N
a
b
+
N
O
S
O
NH2
7
4
5
6
O
N
O
c
N
S
O
N
H
N
H
8
O
N
O
d, e
S
S
N
N
NH2
H
9
O
O
N
S
f
N
NCS
10
Scheme 1. Reagents and conditions: (a) p-nitrophenol carbonate Wang resin (4)/2-aminobenzothiazole (5)/DIEA/60 °C; (b) p-aminophenylethanol/ADDP/Ph3P; (c) benzoyl
isothiocyanate; (d) Fmoc isothiocyanate; (e) piperidine; (f) di-2-pyridylthionocarbanate.
8
a, b, c
d, e, c
R2
H
N
H
N
R
S
S
N
O
N
O
R1
N
N
O
N
H
N
H
N
N
11
R1 = H; R2 = Et, Ph
R
12
R = Me, i-Pr, i-But, c-hexMe, Bn
1 = Ph; R2 = Me
Scheme 2. Reagents: (a) a-amino alcohols/EDCI/TEA; (b) Ph3P/TMAD; (c) TFA cleavage; (d) a-amino acid methyl esters/DCI/TEA; (e) NaOEt.

Z.-Y. Sun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6801–6805
6803
9
a, b
c, b
R1
R
H
N
H
N
S
S
S
S
R2
O
N
N
N
N
N
H
N
H
13
R1 = H;
14
R2 = H, Me, Et, t-But, Ph
R = H, Et, n-Pr, n-But, Ph
R1 = EtOCO; R2 = Me, CF3
R1 = R2 = Me, Ph
Scheme 3. Reagents: (a) a-bromoketones/pyridine; (b) TFA cleavage; (c) a-bromoesters/pyridine.
EtOH to give N-benzoyl imidazolones (12) after TFA cleavage
(Scheme 2). Reaction of resin bound thiourea 9 with -bromoke-
tones and -bromoesters led to the corresponding thiazoles (13)
and 4-thiazolones (14) after cleavage and purification (Scheme 3).
Resin bound isothiocyanate 10 or 15, obtained with different
aminobenzothiazoles and aminophenethyl alcohols, were con-
isothiourea in 1a (data not shown). The substitution pattern of the
phenethyl group is important for thiazolines (20, 21) and dihydro-
thiazines (23, 24) with both meta- and ortho-positions on the phe-
nyl group giving low NPY Y1 binding affinity in contrast to the
corresponding para-substituted analogs 1a and 22a (Table 1). Com-
pounds with five or six-member cyclic thioisourea scaffolds were
found to have comparable NPY Y1 binding affinity (22a, Ki
221 nM vs 1a, Ki 248 nM in Table 1). The dihydrothiazine core is
a novel pharmacophore having NPY Y1 activity.
The SAR for the substitutions on the aminothiazole group is
shown in Table 1. In the compounds with a thiazoline ring, all sub-
stitutions investigated on the aminothiazole seem to reduce the
activity, for example, 1b through 1j (4–24-fold). Substitution on
the aminobenzothiazole seems to be much more tolerated in the
six-membered dihydrothiazine ring series with C6-methyl (22d),
C6-fluoro (22f), C6-chloro (22g), and C5–C6 dichloro (22j) having
comparable Ki range (318–616 nM). However, large substituents
at C6-position reduced the activity, that is, methoxy (22e), nitro
(22h) and methylsulfonyl (22i) groups.
a
a
verted to hydroxyalkyl thioureas upon treatment of a- and b-ami-
noalcohols. These resin bound thioureas were cleaved from resin
with 20% TFA followed by intramoleclular cyclization in neat TFA
by heating13 to form the corresponding cyclic isothioureas 16
and 17 (Scheme 4).16
Finally, we replaced the cyclic isothiourea group with cyclic
amidines as isosteres (19a, 19b), which have an estimated pKa
range of ꢀ8.17 These two compounds were accomplished by con-
densations of intermediate 7a with 18a and 18b (Scheme 5).
All compounds were screened for NPY Y1 receptor binding affin-
ity18 and the Ki values or % inh. at 2
lg/mL are shown in Tables 1
and 2. Imidazolines (11), imidazolones (12), thiazoles (13) and 4-
thiazolones (14) were found to be poor replacements for the cyclic
O
O
N
S
NCS
R3
N
15
R2
R1
R5
N
a, b, c
d, b, c
R5
R4'
R4
H
N
S
H
N
S
R4'
R4
S
S
N
N
H
NH
R3
N
N
R3
R2
R1
R2
R1
16
17
Scheme 4. Reagents and conditions: (a)
a-aminoalcohols; (b) 20% TFA cleavage; (c) neat TFA/heat; (d) b-aminoaminoalcohos.
H
N
n
S
H
H
N
N
O
S
a
N
+
N
H
N
N
NH2
n
7a
18a, n = 0
18b, n = 1
19a, n = 0
19b, n = 1
Scheme 5. Reagents and conditions: TiCl4/toluene/DIEA/heat.

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Z.-Y. Sun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6801–6805
Table 1
NPY Y1 receptor binding affinity of the para-, meta- and ortho-substituted five- and six-member cyclic isothiourea analogs with substituted benzothiazole moieties
H
N
H
N
S
S
1
1
2
2
S
S
N
N
H
NH
N
6
6
R3
R3
N
N3
3
4
5
4
5
R2
R1
R2
R1
1: para-
20: meta-
21: ortho-
22: para-
23: meta-
24: ortho-
Compound
R1
R2
R3
Kia (nM)
%Inhb
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
Me
MeO
F
Cl
O2N
MeO2S
Cl
H
H
H
H
H
Me
MeO
F
248
Me
MeO
H
H
H
H
H
H
H
H
H
H
Me
MeO
H
H
H
3460
3990
1060
1670
1120
1240
5250
5920
3640
1j
20
21
27
27
22a
22b
22c
22d
22e
22f
22g
22h
22i
22j
23
221
77
71
616
1250
318
H
H
H
H
H
H
Cl
342
O2N
MeO2S
Cl
H
H
3300
5200
316
34
24
24
a
NPY Y1 receptor binding affinity Ki values were determined as described in Ref. 18.
Percentage inhibition at 2 lg/mL concentration of testing compounds.
b
Table 2
NPY Y1 receptor binding affinity of the five- and six-membered cyclic isothiourea analogs with substituted aminothiazole/aminothiazine moieties
R5
R5
H
N
H
N
6"
5'
N
S
R4'
R4
1'
S
5"
R4'
1"
S
S
2'
N
R4
N
4'
N
2"
N
H
4"
N
H
3"
3'
25
26
0
Compound
R4
R4
R5
Kia (nM)
%Inhb
25a
25b
25c
25d
25e
25f
25g
25h
25i
Me
Et
H
H
H
H
H
H
H
H
482
379
153
n-Pr/Hc
n-Bu/Hc
i-Bu
69
712
175
H
H
H
Me
OMe
Ph
50
53
Ph
H
H
H
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
0
61
2550
25j
H
17
26a
26b
26c
26d
26e
26f
26g
26h
26i
Me/Hc
i-Pr/Hc
i-Bu/Hc
PhEt/Hc
62
195
332
55
c-Hex/Hc
Ph/Hc
p-OMePh/Hc
p-ClPh/Hc
m-ClPh/Hc
o-ClPh/Hc
Ph
55
122
107
956
H
26j
26k
26l
30
464
H
H
a
b
c
NPY Y1 receptor binding affinity Ki values were determined as described in Ref. 18.
Percentage inhibition at 2
Racemate.
lg/mL concentration of testing compounds.

Z.-Y. Sun et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6801–6805
6805
Table 3
Supplementary data
Functional data and selectivity of 1a and 26k
a
Kb (nM)
Kib (nM)
NPY Y4
Supplementary data (competition binding curves: (1) Inhibition
of radioligand binding by NPY Y1 receptor antagonists 1a and 26k
and (2) Inhibition of NPY functional activity by NPY Y1 receptor
antagonists 1a and 26k) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2009.09.048.
NPY Y1
NPY Y2
NPY Y5
1a
26k
58.5
15.4
248
30
>10,000
>10,000
>10,000
>10,000
>10,000
5978
a
Functional assay Kb values were determined as described in Ref. 19.
b
Binding affinity Ki values for NPY Y1, Y2, Y4, and Y5 receptors were determined
References and notes
as described in Ref. 18 using the cells expressing the human NPY Y1, Y2, Y4 and Y5
receptors, respectively.
1. Tatemoto, K.; Carlquist, M.; Mutt, V. Nature 1982, 296, 659.
2. For reviews, see: (a) Stamford, A. W.; Parker, E. M.. In Ann. Rep. Med. Chem.;
Academic Press, 1999; Vol. 34, p 31; (b) Dumont, Y.; Quirion, R. In Handbook of
Biologically Active Peptides; Elsevier, 2006; p 683.
Substituent effects on the thiazoline and dihydrothiazine rings
are shown in Table 2. At the C40 position of the five membered
thiazoline series (25), small aliphatic groups, such as methyl
(25a) or ethyl (25b), and aromatic groups, such as phenyl (25f),
were tolerated with a range of Ki 153–379 nM, comparable to 1a
(Ki 248 nM). In contrast, the C50 substitutions, that is, methyl
(25h), methoxy (25i) and phenyl (25j), all had much reduced
NPY Y1 binding affinity. The SAR trend in the six-membered dihy-
drothiazine series (26) was quite different. Substitutions at C400 po-
sition, such as methyl (26a), isopropyl (26b), isobutyl (26c) and
phenyl (26f), all significantly enhanced the NPY Y1 binding affinity
by approximately 4–5-folds with Ki values reaching a range from
50 nM to 60 nM. The substitutions on the 400-phenyl group on the
dihydrothiazine were further investigated. The ortho substitution
on the C400-phenyl represented by the o-chloro analog 26j had a
ꢀ10-fold decrease in activity with Ki 956 nM compared to its cor-
responding p- and m-chloro isomers 26h (122 nM) and 26i
(107 nM). Eudismic ratio of both thiazolines and dihydrothiazines
were found to be quite high (>15-fold) with R-enantiomer of C40-
phenyl thiazoline 25f (Ki 175 nM) and S-enantiomer of C400-phenyl
dihydrothiazine 26k (Ki 30 nM) being the more active enantiomers.
Functional antagonism and selectivity against other NPY subtype
receptors were further evaluated for compound 26k. Functionally,
26k was an NPY Y1 antagonist, blocking the NPY mediated inhibi-
tion of the forskolin-stimulated cAMP accumulation in HEK 293
cells with a Kb of 15.4 nM19 (Table 3). For comparison, the Kb for
1a was 58.5 nM. Compound 26k was highly selective against NPY
Y2, Y4, and Y5 receptors. Its Ki values for the Y2 and Y4 receptors
3. Blomqvist, A. G.; Herzog, H. Trends Neurol. Sci. 1997, 20, 294.
4. (a) Kanatani, A.; Ito, J.; Ishihara, A.; Iwaasa, H.; Fukuroda, T.; Fukami, T.; MacNeil,
D. J.; Van der Ploeg, L.; Ihara, M. Reg. Peptides 1998, 75–76, 409; (b) Mullins, D.;
Kirby, D.; Hwa, J.; Guzzi, M.; Rivier, J.; Parker, E. Mol. Pharm. 2001, 60, 534.
5. Rudolf, K.; Eberlein, W.; Engel, W.; Wieland, H. A.; Willim, K. D.; Entzeroth, A.;
Wienen, W.; Beck-Sickinger, A. G.; Doods, H. N. Eur. J. Pharmacol. 1994, 271, R11.
6. Poindexter, G. S.; Bruce, M. A.; LeBoulluec, K. L.; Monkovic, I.; Martin, S. W.;
Parker, E. M.; Iben, L. G.; McGovern, R. T.; Ortiz, A. A.; Stanley, J. A.; Mattson, G. K.;
Kozlowski, M.; Arcuri, M.; Antal-Zimanyi, I. Bioorg. Med. Chem. Lett. 2002, 12, 379.
7. Hipskind, P. A.; Lobb, K. L.; Nixon, J. A.; Britton, T. C.; Bruns, R. F.; Catlow, J.;
Dieckman-McGinty, D. K.; Gackenheimer, S. L.; Gitter, B. D.; Iyengar, S.;
Schober, D. A.; Simmons, R. M. A.; Swanson, S.; Zarrinmayeh, H.; Zimmerman,
D. M.; Gehlert, D. R. J. Med. Chem. 1997, 40, 3712.
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Schober, D. A.; Gackenheimer, S. L.; Bruns, R. F.; Hipskind, P. A.; Britton, T. C.;
Cantrell, B. E.; Gehlert, D. R. J. Med. Chem. 1998, 41, 2709.
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A.; Carpino, P. A. WO Patent 2001023387, 2001.
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12. Fabio, R. D.; Giovannini, R.; Bertani, B.; Borriello, M.; Bozzoli, A.; Donati, D.;
Falchi, A.; Ghirlanda, D.; Leslie, C. P.; Pecunioso, A.; Rumboldt, G.; Spada, S.
Bioorg. Med. Chem. Lett. 2006, 16, 1749.
13. Synthesis of 26k:
A mixture of 2-aminobenzothiazole (5, 10 equiv), p-
nitrophenol carbonate Wang resin (4, 1 equiv) and DIEA (6 equiv) in DMF
was agitated at 60 °C overnight, followed by treatment of methanol/DBU to cap
the unreacted resin as methyl carbonate, to give 6. This resin bound 6 was
treated with aminophenylethanol, PPh3 and ADDP (5 equiv each) in DCM/THF
(1:1) at room temperature overnight to give resin bound 7, which
subsequently reacted with di-2-pyridylthionocarbonate (6 equiv) to give 10
(Scheme 1). This resin bound 10 (300 mg) reacted with (S)-3-amino-3-phenyl-
1-propanol (5 equiv) in 2 mL DCM at room temperature overnight followed by
cleavage with 20% TFA in DCM. The resulting material was treated with neat
TFA at 75 °C overnight to give 26k after purification. 1H NMR (CDCl3, ppm), d
7.59–7.55 (d, 1H), 7.52–7.48 (d, 1H), 7.42–7.35 (m, 4H), 7.34–7.25 (m, 2H),
7.18–7.11 (m, 4H), 7.09–7.05 (m, 1H), 4.69–4.62 (m, 1H), 3.70–3.60 (m, 2H),
3.12–3.04 (m, 1H), 2.98–2.88 (m, 3H), 2.37–2.26 (m, 1H), 2.05–1.92 (m, 1H).
ES-LCMS, m/z = 445 [M+H]+.
were >10 lM, and for the Y5 receptor was 6 lM (Table 3).
The steep SAR observed on the cyclic isothiourea is notable. The
aminothiazole (13, R1, R2 = H) as a replacement of the thiazoline
led to complete loss of NPY1 binding affinity, suggesting that a
pKa difference may play a role in activity. We then explored more
basic six- and seven-membered cyclic amidines (pKa ꢀ8)17 19a and
19b (Scheme 5) as isosteres of cyclic isothioureas (pKa ꢀ7)20 thiaz-
oline 25f and dihydrothiazine 26k. However, they showed much
14. Wilson, M. W.; Hernandez, A. S.; Calvet, A. P.; Hodges, J. C. Mol. Divers. 1998, 3, 95.
15. Poss, M. A.; Iwanowicz, E.; Reid, J. A.; Lin, J.; Gu, Z. Tetrahedron Lett. 1992, 33,
5933.
16. Apparently, the hydroxyl group is converted to the TFA ester which was
reactive enough to be displaced by the thiocarbonyl group of the thiourea.
17. Oszczapowicz, J.; Kuminska, M. J. Chem. Soc., Perkin Trans. 2 1994, 103.
18. Human NPY receptor binding assay. Membranes (5–10
expressing the one of the human NPY receptors were incubated with 0.2 nM
125I] porcine PYY (Y1, Y2 and Y5) or 0.2 nM [125I] human PP (Y4) and various
lg) from CHO-K1 cells
lower activity with Ki 2.9 lM and 4.6 lM, respectively, despite
[
their structural similarities and their close presentations of the
electron density and H-bonding donor/acceptors. Similar scaffolds
with further reduced basicity would be of high interest to further
probe the SAR.
concentrations of antagonist in a buffer containing 50 mM HEPES, pH 7.2,
2.5 mM CaCl2, 1 mM MgCl2 and 0.1% bovine serum albumen for 90 min at
30 °C. Non-specific binding was defined as binding in the presence of 1 lM
human/rat NPY. The reaction mixtures were filtered through Millipore MACF
glass fiber filter plates pre-soaked in 0.5% polyethyleneimine. Filters were
In conclusion, we have identified a series of novel NPY Y1 antago-
nists that bears a cyclicisothiourea group. Multiple efforts inreplace-
ment of this group were not successful. Further substitutions on the
isothiourea group led to identification of 4S-phenyl-dihydro[1,3]thi-
azine compound (26k) as the NPY Y1 antagonist having the best po-
tency with Ki 30 nM. This antagonist may serve as an important tool
for studying the biological functions of NPY Y1 receptor.
washed twice with 150
filter-associated radioactivity was measured in
scintillation counter. The Ki values were calculated using the Cheng–Prussof
equation.
l
l of Dulbecco’s phosphate-buffered saline (4 °C), and
a
Packard TopCount
19. The functional activity of the compounds was determined using a cAMP assay
as described previously.4b Briefly, HEK 293 cells expressing the human NPY Y1
receptor were plated at 1.5 ꢁ 104 cells/well in 24-well dishes. At confluence,
the cells were washed with Hank’s balanced salt solution (HBSS) and incubated
for 20 min in HBSS supplemented with 4 mM MgCl2, 0.2% bovine serum
albumin, 3-isobutyl-1-methyl xanthine (1 mM) and antagonist (1
lM).
Forskolin (2.5 M) and various concentrations of NPY were then added, and
l
Acknowledgment
the incubation continued for 10 min. Cyclic AMP was extracted from the cell
layer using ethanol and measured using a radioimmunoassay (Perkin–Elmer
Flash Plate). Kb values were determined using the Schild equation.
Assistance from Mr. Robert Novotny for the LCMS analysis is
gratefully acknowledged.
20. Dippy, J. F. J.; Hughes, S. R. C.; Rozanski, A. J. Chem. Soc. 1959, 2492.