[35S]GTPγS binding studies of amphiphilic drugs-activated Gi proteins: A caveat

Article abstract of DOI:10.1016/j.bmcl.2009.02.097

This paper documents a serious problem met during the testing of Gi protein-activating properties of a new series of synthetic compounds by measuring the induced binding of [³⁵S]GTPγS to different subtypes of Gi protein. The problem arose from the strong affinity between [³⁵S]GTPγS and the tested compounds, that are characterized by several (2-4) positive charges and high lipophilicity. Apparently, such affinity yields insoluble, labelled complexes that, also in the absence of Gi protein, are retained on the filters and give rise to false positive results.

Full text of DOI:10.1016/j.bmcl.2009.02.097

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2224–2229
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
[35S]GTP
cS binding studies of amphiphilic drugs-activated Gi proteins: A caveat
b
a
a
a
Dina Manetti a, , Lorenzo Di Cesare Mannelli , Silvia Dei , Luca Guandalini , Elisabetta Martini ,
*
Martina Banchelli c, Carla Ghelardini b
a Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente Attivi (HeteroBioLab), Dipartimento di Scienze Farmaceutiche, Università di Firenze, Via U. Schiff 6,
50019 Sesto Fiorentino (FI), Italy
b Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy
c Dipartimento di Chimica e CSGI, Università di Firenze, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper documents a serious problem met during the testing of Gi protein-activating properties of a
new series of synthetic compounds by measuring the induced binding of [35S]GTP
c
S to different subtypes
Received 14 January 2009
Revised 24 February 2009
Accepted 25 February 2009
Available online 28 February 2009
of Gi protein. The problem arose from the strong affinity between [35S]GTP
cS and the tested compounds,
that are characterized by several (2–4) positive charges and high lipophilicity. Apparently, such affinity
yields insoluble, labelled complexes that, also in the absence of Gi protein, are retained on the filters
and give rise to false positive results.
Keywords:
Amphiphilic drugs
Gi protein
Ó 2009 Elsevier Ltd. All rights reserved.
Binding studies
A receptor-independent modulation of the heterotrimeric G
proteins is an intriguing purpose. A selective, single-subunit
modulator represents a suitable means to intervene in the com-
plex intracellular pathways. Direct modulation could be useful in
those pathological conditions where a G protein involvement is
already demonstrated. Indeed, altered G proteins are involved
For this purpose we chose liposomes as the best biodegradable
or biocompatible drug carriers.14 Aiming to improve the potency
and selectivity of previously studied compounds and to establish
sounder structure–activity relationships, we have continued our
research synthesizing and studying the compounds shown in
Table 1.
in several pathologic conditions: mutations in the G
a
inhibitory
4-Aminopiperidines 1–10 were prepared according to the pro-
cedure shown in Scheme 1. Commercially available 4-piperidone
hydrochloride monohydrate was treated with di-tert-butyl dicar-
bonate and anhydrous NEt3, then the intermediate 33 was trans-
formed into 34–40 by reductive amination15 with the appropriate
alkylamine. After deprotection with HCl or with trifluoroacetic
subunit (G i) codifying genes have been associated with tu-
a
mours1–3 and there is increasing evidence for implications in
infections, inflammations, neurological and cardiovascular
diseases and endocrine disorders.4,5 Moreover, a hypofunctional-
ity of Gai in lymphocytes of cephalalgic and fibromialgic patients
was demonstrated.6,7 Among drugs known to modulate G pro-
teins in a receptor-independent manner,8–10 a novel series of
low molecular weight derivatives were found to be able to stim-
**
acid (see experimental part in the Supplementary data ) these
compounds gave 1–7. 4-Pentadecilamine piperidine (BC5), pre-
pared by the same method,11 was alkylated with bromoethyl-
amine hydrobromide to obtain 8. Compounds 9 and 10 were
obtained from BC5 and 8, respectively, in a three-step procedure
ulate the Gai-protein signalling pathway in human lymphocytes
and to activate isolated recombinant Gai proteins.11,12 Among
these derivatives, a 4-aminopiperidine derivative named BC5 is
able to modulate cAMP levels in a recombinant system reconsti-
acylating with Na-Boc-Ne-trifluoroacetyl-L-lysine to yield 41 and
42, and deprotecting the Boc- and trifluoroacetyl groups with tri-
fluoroacetic acid and with K2CO3, respectively. Compound 11 was
synthesized in a similar way (Scheme 2): 1-pentadecylpiperidin-
tuted with the isoform 1 of Gai subunit (Gai1) and the intracel-
lular fragments of adenylate cyclase.13 Moreover, to improve
screening accuracy and enhance efficacy, and to reduce the
toxicity of therapeutics, we proceeded with the reconstitution
4-ylamine11 was acylated with N
a-Boc-Ne-trifluoroacetyl-L-lysine
and then deprotected. 4,4’-Bipiperidines 12–23 were synthesized
as shown in Scheme 3, starting from commercially available 4,40-
bipiperidine dihydrochloride which was treated with 10% NaOH,
reacted with BOC-ON [2-(tert-butoxycarbonyloxyimino)-2-phen-
ylacetonitrile], then treated with the suitable bromoalkyl deriva-
tives and NEt3 as a scavenger, and finally deprotected to give
of the
G
protein molecules in
a
phospholipid bi-layer.
* Corresponding author. Tel.: +39 055 4573688; fax: +39 055 4573780.
E-mail address: dina.manetti@unifi.it (D. Manetti).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.097

D. Manetti et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2224–2229
2225
Table 1
Chemical and physical characteristics of final derivatives 1–32
NHR1
NHR1
NHR1
Ν
R2
R1
N
N
N
R2
N
N
D
A
B
C
R2
H
N
Structure
R1
R2
Salt (mp °C)a
Analysis (salt)
1
2
3
4
5
6
7
8
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
C
C
C
C
C
D
D
D
D
(CH2)4CH3
(CH2)6CH3
(CH2)8CH3
(CH2)4Ph
(CH2CH2O)3CH2CH3
(CH2CH2O)4CH2CH3
(CH2CH2O)5CH2CH3
CH2(CH2)13CH3
H
H
H
H
H
H
H
2HCl (309–310)
2HCl (307–308)
2HCl (295–297)
2HCl (277–281)
2HCl (low melting)
2HCl (196–200)
2HCl (220–225)
3HCl (201–205)
3HCl (196–200)
4HCl (202–206)
3HCl (246–249)
2HCl (269–273)
2HCl (270–271)
2HCl (280–285)
2HCl (275–285)
2HCl (low melting)
2HCl (214–218)
2HCl (220–224)
3HCl (276–280)
2HCl (223–225)
3HCl (189–192)
4HCl (215–218)
2HCl (237–240)
3HCl (low melting)
3HCl (238–242)
3HCl (236–240)
3HCl (225–230)
3HCl (low melting)
2HCl (low melting)
2HCl (220–222)
2HCl (low melting)
2HCl (218–220)
C10H24Cl2N2
C12H28Cl2N2
C14H32Cl2N2
C15H26Cl2N2
C13H30Cl2N2O3
C15H34Cl2N2O4
C17H38Cl2N2O5
C22H50Cl3N3
C26H57Cl3N4O
C28H63Cl4N5O
C26H57Cl3N4O
C15H32Cl2N2
C17H36Cl2N2
C19H40Cl2N2
C20H34Cl2N2
C18H38Cl2N2O3
C20H42Cl2N2O4
C22H46Cl2N2O5
C27H58Cl3N3
C27H55Cl2N3O
C31H65Cl3N4O
C33H71Cl4N5O
C31H62Cl2N4O2
C9H24Cl3N3
CH2CH2NH2
-Lysine
CH2CH2NH-
9
CH2(CH2)13CH3
CH2(CH2)13CH3
L
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
L
-lysine
L-Lysine
CH2(CH2)13CH3
H
H
H
H
H
H
H
(CH2)4CH3
(CH2)6CH3
(CH2)8CH3
(CH2)4Ph
(CH2CH2O)3CH2CH3
(CH2CH2O)4CH2CH3
(CH2CH2O)5CH2CH3
CH2(CH2)13CH3
CO(CH2)13CH3
CH2(CH2)13CH3
CH2(CH2)13CH3
CO(CH2)13CH3
(CH2)4CH3
(CH2)6CH3
(CH2)8CH3
(CH2)4Ph
(CH2CH2O)3CH2CH3
(CH2)4CH3
(CH2)4Ph
(CH2CH2O)3CH2CH3
(CH2)14CH3
CH2CH2NH2
CH2CH2NH2
L
-Lysine
CH2CH2NH-
-Lysine
L
-lysine
L
—
—
—
—
—
H
H
H
C11H28Cl3N3
C13H32Cl3N3
C14H26Cl3N3
C12H30Cl3N3O3
C11H26Cl2N2
C16H28Cl2N2
C14H32Cl2N2O3
C24H51Cl2N3O
CH2CH2NHCHO
a
From absolute ethanol/anhydrous diethyl ether.
12–18. In the same manner the already described N-pentadecylb-
ipiperidine 5511 or N-pentadecanoylbipiperidine 5611 were ob-
tained; alkylation of these intermediates with bromoethylamine
hydrobromide yielded 19 and 20, while acylation of 55, 19 and
and positively charged molecules, belong to the class of
surface-active drugs. Understandably, the formulation and the
screening of surface-active drugs represents a critical issue. It
is well known that amphiphilic drugs can self-associate and bind
to plasma membrane, causing disruption and solubilization of
the lipid bi-layer, similarly to common detergents. As a matter
of fact, we were prepared to face the problems related to their
tendency to self-assembly that could induce aspecific effects
and therefore compromise the reliability of the functional tests
56 with
Na-Boc-Ne-trifluoroacetyl-L-lysine and subsequent
deprotections yielded compounds 21–23, respectively. Pipera-
zines 24–28 were synthesized as shown in Scheme 4. 1-Amino-
4-benzylpiperazine 6316 was alkylated with the appropriate bro-
moalkane or bromoethoxyethane and then debenzylated with
HCOONH4 and 10% Pd/C in MeOH to give compounds 24–28. 4-
Methylalkylaminopiperidines 29–32 were prepared as reported
in Scheme 5. N-benzyl isonipecotic acid 6911 was transformed
into the corresponding amides 70–72 using ethyl chloroformate,
NEt3 and the appropriate amine. After reduction with borane di-
methyl sulphide complex, compounds 73–75 were reduced with
HCOONH4 and 10% Pd/C in MeOH to give derivatives 29–31. N-
(Piperidin-4-ylmethyl)pentadecan-1-amine 76,11 was alkylated
with bromoethylamine hydrobromide to obtain compound 32.
According to previously reported protocols,11–13 we evaluated
the G-protein activation activity of our compounds by measuring
of GTPcS binding. However, the cause that eventually aborted
our efforts to evaluate the Gi-activating activity of these com-
pounds was unexpected and apparently independent from their
tendency to self-aggregate. After a few confusing results, we
soon realized that in most cases radioactivity was present on
the filters even in the absence of Gi protein, indicating that
the molecules tested were able to bind with [35S]GTP
cS forming
insoluble complexes that were retained on the filters. Of course,
this fact rendered the results of the test largely unreliable. In Ta-
ble 2 some illustrative data obtained both in the presence and
absence of Gi protein of selected compounds of the series are re-
ported, in comparison with our standard derivative BC5 (data are
reported also for the alphao subunit). Other compounds of the
the influence of these latter on [35S]GTP
ent subtypes of Gi protein. These compounds, being lipophilic
cS binding to the differ-

2226
D. Manetti et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2224–2229
34:R1=(CH2)4CH3
NHR1
35:R1=(CH2)6CH3
O
N
HO
OH
36:R1=(CH2)8CH3
37:R1=(CH2)4Ph
a
b
38:R1=(CH2CH2O)3CH2CH3
39:R1=(CH2CH2O)4CH2CH3
40:R1=(CH2CH2O)5CH2CH3
N
N
HCl
33
OC(CH3)3
H
O
O
OC(CH3)3
c
1:R1=(CH2)4CH3
NH(CH2)14CH3
d (on BC5)
NHR1
2:R1=(CH2)6CH3
3:R1=(CH2)8CH3
4:R1=(CH2)4Ph
5:R1=(CH2CH2O)3CH2CH3
6:R1=(CH2CH2O)4CH2CH3
7:R1=(CH2CH2O)5CH2CH3
BC5: R1=(CH2)14CH3
8
N
N
H
CH2CH2NH2
e
e (on BC5)
NH(CH2)14CH3
NH(CH2)14CH3
NH(CH2)14CH3
g
f
N
N
N
R3
R1
R4
9:R2= lysine
43:R4= Nε-
41:R3= Nα-Boc-Nε-
trifluoroacetyl-L-lysine
42:R3= CH2CH2NH(Nα-
Boc-Nε-trifluoroacetyl)-L-
lysine
10:R2= CH2CH2NH-L-lysine
trifluoroacetyl-L-lysine
44:R4=
CH2CH2NH(Nε-
trifluoroacetyl)-L-
lysine
Scheme 1. Reagents and conditions: (a) (tBuOCO)2O, NEt3 anhyd; (b) NH2X, (iPrO)4Ti, NaBH3CN, 25–81% yields; (c) HCl 6 M or CF3COOH (see experimental part), 67–100%
yields; (d) BrCH2CH2NH2 HBr, K2CO3, 40% yield; (e) N
a
-Boc-N
e
-trifluoroacetyl-
L
-lysine, 1,10-carbonyldiimidazole, 42% yields; (f) CF3COOH, 83–93% yields; (g) K2CO3, 78–96%
yields.
series either gave binding values close to basal (no Gi activation;
compounds 1–7, 12–18, 24–31) or they behaved like the com-
pounds reported in Table 2 (compounds 8–10, 21, 23).
32). On the other hand, the solubility of the salts in the test
medium can change according to the structure and dose of
tested compounds; this may explain why in many cases (BC5
in Table 2 and all the compounds reported previously)11,12 we
either did not notice the problem or were able to overcome it
A reasonable explanation for this result is that the positively
multi-charged, hydrophobic molecules of our set combine with
the negatively charged [35S]GTP
cS to give salts that are insoluble
at suitable doses.
in the medium of the assay. The positive correlation between the
concentration of the compounds and the radioactivity found on
the filter supports this explanation (see for instance compound
It must be emphasized that this case is somehow different
from the micelle aggregation proposed in the recent litera-
ture17,18 to explain ‘frequent hits’ and ‘promiscuous inhibitors’
during high throughput screening, that is, the formation of mi-
celle aggregates that interact unselectively with biologically ac-
tive molecules inhibiting them. However, micelle aggregates
could play a role even in this case, contributing to the poor sol-
NHR3
NH2
a
ubility of the salt between the tested compound and [35S]GTP
cS.
45:R3= Nα-Boc-Nε-
To clarify this point, we checked the tendency of reference com-
pound BC5, and 8 and 22 to give micelle aggregates by monitor-
ing the changes produced on air/water surface tension. As can be
seen in Figure 1, the compounds show a critical micelle concen-
tration (cmc) around 1 ꢀ 10ꢁ4 M (1.0 ꢀ 10ꢁ4, 1.41 ꢀ 10ꢁ4 and
1.44 ꢀ 10ꢁ4, respectively). These values suggest that, at lower
concentrations, self-aggregation does not critically contribute to
trifluoroacetyl-L-lysine
N
N
(CH2)14CH3
(CH2)14CH3
b
NHR1
NHR4
the insolubility of the complex with [35S]GTP
cS allowing, in
c
the absence of other interferences, a reliable evaluation of their
binding to the protein. It was expected that, at cmc or higher
concentrations, the micelle formation would aggravate the prob-
lem contributing to the failure of the assay: this indeed proved
to be the case, as verified experimentally for BC5 (see Table 2).
Therefore, the anomalous behaviour found for the compounds
reported in Table 2 seems to be due exclusively to the formation
N
N
(CH2)14CH3
(CH2)14CH3
46:R4= Nε-
trifluoroacetyl-
L-lysine
11:R1= lysine
of insoluble salts between [35S]GTP
pounds tested.
cS and some of the com-
Scheme 2. Reagents and conditions: (a) N
carbonyldiimidazole, 76% yield; (b) CF3COOH, 95% yield; (c) K2CO3, 58% yield.
a-Boc-Ne-trifluoroacetyl-L
-lysine, 1,10-

D. Manetti et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2224–2229
2227
a
HN
NH
BocN
NR1
b
BocN
NH
HCl
HCl
48:R1=(CH2)4CH3
47
c
49:R1=(CH2)6CH3
50:R1=(CH2)8CH3
51:R1=(CH2)4Ph
NR1
HN
52:R1=(CH2CH2O)3CH2CH3
53:R1=(CH2CH2O)4CH2CH3
54:R1=(CH2CH2O)5CH2CH3
12:R1=(CH2)4CH3
13:R1=(CH2)6CH3
14:R1=(CH2)8CH3
15:R1=(CH2)4Ph
d (on 55 and 56)
e (on 55 and 56)
16:R1=(CH2CH2O)3CH2CH3
17:R1=(CH2CH2O)4CH2CH3
18:R1=(CH2CH2O)5CH2CH3
55:R1=(CH2)14CH3
56:R1=CO(CH2)13CH3
N
R1
R3
N
R1
H2NH2CH2C
N
N
57:R1=(CH2)14CH3; R3= Nα-Boc-
Nε-trifluoroacetyl-L-lysine
58:R1=(CH2)14CH3;
19:R1=(CH2)14CH3
20:R1=CO(CH2)13CH3
e (on 19)
f
R3=CH2CH2NH(Nα-Boc-Nε-
trifluoroacetyl)-L-lysine
59:R1=CO(CH2)13CH3; R3= Nα-
Boc-Nε-trifluoroacetyl-L-lysine
R2
N
R1
g
N
R4
N
R1
N
21:R1=(CH2)14CH3;R2=lysine
60:R1=(CH2)14CH3; R4=Nε-trifluoroacetyl-
L-lysine
22:R1=(CH2)14CH3; R2=CH2CH2NH-L-lysine
23:R1=CO(CH2)13CH3; R2= L-lysine
61:R1=(CH2)14CH; R4=CH2CH2NH(Nε-
trifluoroacetyl)-L-lysine
62:R1=CO(CH2)13CH3; R4= Nε-
trifluoroacetyl-L-lysine
Scheme 3. Reagents and conditions: (a) 10% NaOH, BOC-ON; (b) bromoalkane or bromo-ethoxyethane, NEt3, 10–47% yields; (c) HCl 6 M or CF3COOH (see experimental part),
71–100% yields; (d) BrCH2CH2NH2 HBr, K2CO3, 32–34% yields; (e) Na-Boc-Ne-trifluoroacetyl-L
-lysine, 1,10-carbonyldiimidazole, 16–93% yields; (f) CF3COOH, 76–93% yields;
(g) K2CO3, 60–70% yields.
NHR1
N
NHR1
NH2
N
O
O
OH
64:R1=(CH2)4CH3
65:R1=(CH2)6CH3
66:R1=(CH2)8CH3
67:R1=(CH2)4Ph
70:R1=(CH2)4CH3
a
a
71:R1=(CH2)4Ph
N
N
72:R1= (CH2CH2O)3CH2CH3
N
N
69
68:R1=(CH2CH2O)3CH2CH3
CH2Ph
CH2Ph
CH2Ph
b
CH2Ph
63
b
NHR1
24:R1=(CH2)4CH3
25:R1=(CH2)6CH3
26:R1=(CH2)8CH3
27:R1=(CH2)4Ph
NHR1
N
NHR1
73:R1= (CH2)4CH3
c
N
H
74:R1= (CH2)4Ph
28:R1=(CH2CH2O)3CH2CH3
75:R1= (CH2CH2O)3CH2CH3
N
N
H
Scheme 4. Reagents and conditions: (a) BrX, NEt3, 36–85% yields; (b) Pd/C/
HCOONH4, 54–75% yields.
CH2Ph
d (on 76)
NHR1
29:R1=(CH2)4CH3
30:R1=(CH2)4Ph
Obviously, as far as Gi activation properties of our compounds
are concerned, we can only say that the compounds showing
31:R1=(CH2CH2O)3CH2CH3
76:R1=(CH2)14CH3
[
35S]GTP
cS binding close to basal are inactive (see the list re-
N
32:R1= (CH2)14CH3
ported above). Nothing can be inferred about the activating
properties of the compounds listed in Table 2 and of those that
behave similarly. A more reliable assay is necessary to evaluate
their activity. Our results suggest that much care should be ta-
ken when performing binding studies with charged, highly lipo-
philic molecules.
NHCHO
Scheme 5. Reagents and conditions: (a) ClCOOEt, NH2R, 39–68% yields; (b)
(CH3)2SBH3, 36–44% yields; (c) Pd/C/HCOONH4, 24–60% yields; (d) BrCH2CH2NH2
HBr, DMF, 40% yield.

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D. Manetti et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2224–2229
Table 2
GTPcS binding on the alphai1 subunit of G proteins (fmol bound in 15 min at 30 °C)
Basal
Basal
1 ꢀ 10ꢁ4
M
5 ꢀ 10ꢁ5
M
1 ꢀ 10ꢁ5
M
5 ꢀ 10ꢁ6
M
1 ꢀ 10ꢁ6
M
Variationa (%)
ao 82.8 4.75
i1 8.9 0.9
a
11
19
20
22
32
BC5
BC5b
a
With Gi protein
Without protein
184.8 20.2*
301.9 35.2
2304.0 235.9*
567.4 79.1
157.0 14.7*
234.4 22.2
234.4 19.0*
149.4 22.8
102.8 9.8*
176.6 19.8
78.7 13.5*
23.7 5.6*
34.6 4.8
1664
2553
877
With Gi protein
Without protein
11.6 1.2
With Gi protein
Without protein
102.9 10.9*
145.8 9.4
781.0 97.9*
835.8 101.7
980.0 90.1*
901.2 78.8
195.5 10.2*
46.7 12.7
87.0 9.8*
119.8 10.8
275.1 22.1*
192.8 13.2
112.8 16.8*
321.4 24.6
70.6 5.2*
28.6 3.7
636.3 78.6*
248.8 20.1
76.2 13.7*
107.2 16.5
33.3 9.7*
111.7 9.2
69.1 6.9*
133.4 12.2
49.5 5.6*
8.9 2.1
362.8 58*
68.8 8.1
30.3 11.1*
58.3 12.5
With Gi protein
Without protein
19.9 9.6
2991
1167
693
With Gi protein
Without protein
8.9 2.5
With Gi protein
Without protein
194.8 14.1
178.0 10.1
15.4 2.6*
1.2 0.4
With Gi protein
Without protein
1422.5 152
1387.0 178
1395.0 94.7*
319.0 18.1
102.3 10.3
9.2 2.3
668
Variation % in respect to basal at 1 ꢀ 10ꢁ5 M.
b
GTPcS binding on the alphao subunit of G proteins, with and without protein.
*
P < 0.05.
Figure 1. Air/water surface tension measurements.
2. Williamson, E. A.; Ince, P. G.; Harrison, D.; Kendall Taylor, P.; Harris, P. E. Eur. J.
Clin. Invest. 1995, 25, 128.
Acknowledgement
3. Spiegel, A. M.; Weinstein, L. S. Annu. Rev. Med. 2004, 55, 27.
4. Lerman, B. B.; Dong, B.; Stein, K. M.; Markowitz, S. M.; Linden, J.; Catanzaro, D.
F. J. Clin. Invest. 1998, 101, 2862.
We are grateful to Professor Fulvio Gualtieri for helpful
discussion.
5. Melien, O. Methods Mol. Biol. 2007, 361, 119.
6. Galeotti, N.; Ghelardini, C.; Zoppi, M.; Del Bene, E.; Raimondi, L.; Beneforti, E.;
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Supplementary data
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Supplementary data relative to the pharmacological method,
chemistry, yield and chromatographic eluent of intermediates,
chemical characterization of intermediates and final derivatives
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