Semisynthesis and antimicrobial activity of novel guttiferone-A derivatives

Article abstract of DOI:10.1016/j.bmc.2012.02.023

Six derivatives of guttiferone-A (LFQM-79, 80, 81, 82, 113 and 114) were synthesized and evaluated for their antimicrobial activity against the opportunistic or pathogenic fungi Candida albicans (ATCC 09548), Candida glabrata (ATCC 90030), Candida krusei

Full text of DOI:10.1016/j.bmc.2012.02.023

Bioorganic & Medicinal Chemistry 20 (2012) 2713–2720
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Semisynthesis and antimicrobial activity of novel guttiferone-A derivatives
Kris S.T. Dias a, Jaqueline P. Januário a, Jéssica Lopes D’ Dego a, Amanda L.T. Dias b, Marcelo H. dos Santos a,
Ihosvany Camps c, Luiz Felipe L. Coelho b, Claudio Viegas Jr. a,
⇑
a Laboratory of Phytochemistry and Medicinal Chemistry, Institute of Chemistry, Federal University of Alfenas, 37130-000 Alfenas, MG, Brazil
b Laboratory of Microbiology and Immunology, Institute of Biomedical Scienses, Federal University of Alfenas, 37130-000 Alfenas, Brazil
c Laboratory of Computational Modeling, Institute of Exact Scienses, Federal University of Alfenas, 37130-000 Alfenas, MG, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Six derivatives of guttiferone-A (LFQM-79, 80, 81, 82, 113 and 114) were synthesized and evaluated for
their antimicrobial activity against the opportunistic or pathogenic fungi Candida albicans (ATCC 09548),
Candida glabrata (ATCC 90030), Candida krusei (ATCC 6258), Candida parapsilosis (ATCC 69548), Candida
tropicalis (ATCC 750), Cryptococcus neoformans (ATCC 90012), Trichophyton tonsurans, Microsporum gypse-
um and also against the opportunistic and pathogenic Gram-positive bacteria Staphylococcus aureus
(ATCC 6538), Staphylococcus epidermidis (ATCC 12228), Bacillus cereus (ATCC 11778) and Gram-negative
Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC
14028), Proteus mirabilis (ATCC 25933). The antimicrobial activities of derivatives were compared with
guttiferone-A and they presented to be more potent than the original molecule and sometimes greater
than standard drugs established in therapeutics. The current study showed that derivatives of guttifer-
one-A possess potent antimicrobial activity and are relatively non-cytotoxic, which reveal these new
molecules as promising new drug prototype candidates, with innovative structural pattern.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 30 November 2011
Revised 31 January 2012
Accepted 7 February 2012
Available online 18 February 2012
Keywords:
Benzophenones
Guttiferone-A
Antimicrobial activity
1. Introduction
Guttiferone-A (1) is one of the most abundant natural polyisopr-
enylated benzophenone isolated from G. brasiliensis, and many
Infectious diseases represent a critical issue for health and are
the major cause of morbidity and mortality worldwide. Despite
significant progress in human medicine, infectious diseases caused
by microorganisms are still a serious threat to public health. The
impact is even greater in developing countries due to unavailabil-
ity of medicine in all locations, the practice of self-medication and
the emergence of microorganism drug resistance.1
The polyisoprenylated benzophenone derivatives compounds
are a very restricted class of plant natural products, occurring
mainly in the family Guttiferae that excels due to benzophenones
usually replaced by isoprenyl or geranyl subunits, which can be cy-
clized or not. For several polyisoprenylated benzophenones, anti-
microbial, cytotoxic, antiviral, antioxidant and antiinflammatory
activities have been reported.2
The species Garcinia brasiliensis is native to the Amazon region
and is cultivated throughout the Brazilian territory and is known
as ‘bacupari’. It is a tree of medium size that blooms from August
to September and presents a yellow fruit with a white and edible
mucilaginous pulp. In folk medicine, the leaves of G. brasiliensis
are used to treat tumors, inflammation of the urinary tract, arthri-
tis and to relieve pain.3
pharmacological activities have been reported for this metabolite,
including anti-HIV,4 cytotoxic,5 trypanocidal, antiplasmodial,
antioxidant,6 inhibitory activity of cysteine and serine proteases,7
and antimicrobial.8,9 In a continuing search for new antimicrobial
drug candidates, we elected compound 1, that had already showed
antimicrobial activities,8,9 as a model for molecular modifications
aiming the preparation of more potent derivatives and the establish-
ment of a structure–activity relationship and evaluate the contribu-
tions of functional group modifications in the lipophilicity of the
target derivatives. Hydroxyl groups at C-13 and C-14 positions were
focused for chemical modifications based on their higher reactivity
and on previous data10 that indicates the importance of the chelato-
genic system on ring B for biological activity. Thus, in this report we
present the semisynthetic preparation of a series of six new deriva-
tives of natural guttiferone-A (1), isolated from the seeds of G.brasil-
iensis, and the results of their antimicrobial evaluation.
2. Results and discussion
2.1. Semisynthesis of guttiferone-A derivatives
The synthesis of guttiferone-A (1) derivatives LFQM-79 (2),
LFQM-80 (3), LFQM-81 (4), LFQM-82 (5), LFQM-113 (6) and LFQM-
114 (7) are shown in Figure 1. The isolation of starting material was
optimized from the ethyl acetate extract of seeds of G. brasiliensis
⇑
Corresponding author. Tel./fax: +55 35 32991466.
E-mail addresses: cvjviegas@gmail.com, viegas@unifal-mg.edu.br (C. Viegas).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.023

2714
K. S. T. Dias et al. / Bioorg. Med. Chem. 20 (2012) 2713–2720
23
21
19
23
21
37
37
O
O
OH
O
O
OH
19
O
3
5
11
13
15
35
O
11
13
5
15
41
3
35
9
9
1
17
39
1
O
O
O
17
7
39
O
O
O
7
27
25
OH
29
27
25
O
29
41
43
31
23
21
19
31
33
33
LFQM-79 (2)
37
O
OH
LFQM-80 (3)
O
5
11
13
15
3
35
a
9
S
b
1
17
O
O
O
O
7
39
O
27
25
23
O
29
23
21
S
31
c
O
37
LFQM-81 (4)
OH
O
OH
33
19
11
13
5
9
15
3
35
1
17
21
19
HO
O
O
7
37
O
O
27
25
OH
29
d
O
5
9
11
13
15
3
35
31
1
17
33
39
45
O
O
7
Guttiferone-A (1)
27
41
43
25
O
O
29
Cl
f
31
e
47
23
33
51
49
LFQM-82 (5)
21
37
35
O
O
OH
Cl
19
23
O
3
11
15
9
21
19
O
O
OH
13
1
37
17
25
45
39
O
O
O
7
27
41
43
O
S
5
11
13
O
29
15
3
35
9
1
17
31
O
O
7
43 39
41
47
51
O
27
33
25
O
O
29
49
S
LFQM-114 (7)
45
O
31
47
51
33
49
LFQM-113 (6)
Figure 1. Synthesis of guttiferone-A derivatives (2–7). Reagents and conditions: (a) Boc2O/DMAP in CH2Cl2; (b) Ac2O/DMAP in CH2Cl2 (c) MsCl/Et3N in CH2Cl2 (d)
Chlorobenzoylchoride/K2CO3 in acetone; (e) TsCl/Et3N in CH2Cl2; (f) benzoylchoride/K2CO3 in acetone.
affording compound 1 in yield from the seeds (8% yield form ethyl
acetate extract). Complete 1H and 13C NMR data are displayed on
Tables 5 and 6.
Table 1
Calculated lipophilicity of compounds 1–7 expressed as cLogP (oct/wat)
Compound
cLogP (oct/wat)
In order to synthesize compound LFQM-79 (2), guttiferone-A (1)
was reacted with di-tert-butoxycarbonyl anhydride and 4-DMAP in
CH2Cl2. The molecular weight of compound 2 was determined by
HRESI-MS to be 725.4033 [M+Na]+, consistent with a molecular
formula of C43H58O8Na.
Guttiferone-A (1)
LFQM-79 (2)
LFQM-80 (3)
LFQM-81 (4)
LFQM-82 (5)
LFQM-113 (6)
LFQM-114 (7)
9.24
10.82
9.06
5.20
11.11
8.75
Analysis by 1H and 13C NMR, revealed a singlet for 9H at d 1.11,
related to three equivalent methyl groups, and the presence of
three carbon signals at d 140.76, 83.18 and 26.58, attributed to
the carbonyl, quaternary carbon and methyl group, respectively,
confirmed the desired conversion of only one hydroxyl group into
the correspondent di-tert-butoxycarbonyl ester.
11.85
and strong absorption bands at 1766 and 1257 cmÀ1, consistent, to
the presence of an ester group bands. The UV spectrum displayed
Furthermore, the IR spectrum exhibited absorption bands at
absorptions with kmáx (log
the pure compound. Addition of AlCl3 produced absorptions at
e) at 275 nm (3.45), 250 nm (3.46) for
3446 cmÀ1, indicating that there was no substitution of all hydroxyl

K. S. T. Dias et al. / Bioorg. Med. Chem. 20 (2012) 2713–2720
2715
326 nm (3.35), 251 nm (3.41) and 223 nm (3.55). Addition of HCl
The UV spectrum displayed absorptions with kmáx (loge) at
produced absorptions at 328 nm (3.37), 252 (3.42) and 223 nm
(3.59). These shift changes observed in the UV spectra produced
by adding AlCl3 and HCl indicated the presence of chelatogenic hy-
droxyl group, confirming that the reaction does not occurred on the
hydroxyl group at C-4 position. According to the UV data, jointly
with NMR and MS analysis, compound LFQM-79 (2) was deduced
as 14-O-tert-butoxycarbonyl-guttiferone-A.
The reaction of 1 with acetic anhydride and triethylamine in
CH2Cl2 at room temperature gave compound LFQM-80 (3). The
molecular weight of compound 3 was determined by HRESI-MS to
be 709.3709 [M+Na]+, consistent with a molecular formula of
244 nm (4.21) for the pure compound. Addition of AlCl3 led to
absorptions at 468 nm (2.27) and 244 nm (4.18). Addition of HCl
produced absorptions at 468 nm (2.04) and 227 nm (4.12). Again,
these shift changes in the UV absorption produced by adding AlCl3
and HCl revealed that the chelatogenic hydroxyl group at C-4
position was preserved. According to these spectroscopic data,
compound LFQM-82 was confirmed as 13,14-di-chlorobenzoyl-
guttiferone-A (5).
Compound LFQM-113 (6) was obtained by reaction of com-
pound 1 with p-toluenesulfonyl chloride and triethylamine in
CH2Cl2. The molecular weight of compound 6 was determined by
HRESI-MS to be 933.3645 [M+Na]+, consistent with a molecular
formula of C52H62O10S2Na. The formation of tosyl ester was
evidenced in the IR spectrum by the absorption bands at
1749 cmÀ1 and 1246 cmÀ1 characteristic of axial deformations of
C@O of ester and C–O–C@O, respectively. The formation of the to-
syl-guttiferone derivative was also confirmed by 1H and 13C NMR.
In the lower field region of 13C NMR spectrum, it was observed
characteristic signals for tosyl subunit at d 130.42 (C-39), 128.57
(C-40), 129.84 (C-41), 144.05 (C-42), 129.84 (C-43), 128.57
(C-44), 127.87 (C-46), 128.57 (C-47), 129.84 (C-48), 145.77 (C-
49), 129.84 (C-50), 128.57 (C-51) and the methyl carbon attached
to aromatic ring 21.78 (C-45), 21.78 (C-52). The presence of aro-
matic hydrogen signals at d 7.65 (H-40, 44, 47, 51) and 7.29 (H-
41, 43, 48, 50), along with hydrogen signal of the methyl group
bound to the benzene ring at 2.45 (H-45 and 52). The UV spectrum
C42H54O8Na. As observed for all semisynthetic derivatives, most
signals in 1H and 13C NMR spectra of 3 were similar to compound
1, except for an extra signal at d 2.22 (6H, s) in the 1H NMR spectrum
related to the two methyl groups and the signals for two methylic
carbons at d 19.55 and 19.72, and two carbonyl carbons at d
166.45 and 166.75, observed in the 13C NMR spectrum, indicated
that two hydroxyl groups were converted to the correspondent ace-
tate ester groups. In the IR spectrum of compound 3, the absence of a
hydroxyl absorption band at 3267 cmÀ1 and the appearance of an
absorption band at 1778 cmÀ1, a typical stretch of C@O ester, sug-
gested completion of the substitution reaction. The UV spectrum
displayed absorptions with kmáx (loge) at 293 nm (3.84), 248 nm
(3.97) and 202 nm (4.23) for the pure compound. The addition of
AlCl3 produced a shift at 324 nm (3.86), 252 nm (3.97) and
222 nm (4.00). The addition of HCl does not restore the spectrum,
showing absorptions at 327 nm (3.88), 252 nm (3.97) and 223 nm
(4.03). On the basis of these spectroscopic data, compound LFQM-
80 was elucidated as 13,14-di-acetyl-guttiferone-A (3).
displayed absorptions with kmáx (loge) at 484 nm (2.10) and
227 nm (4.35), for the pure compound. Absorptions at 468 nm
(2.13), 312 nm (3.55) and 222 nm (4.36) were produced when
AlCl3 was added. The system could not be regenerated with the
addition of HCl, producing absorptions at 468 nm (1.89) and
313 nm (3.50) that was indicative of the presence of chelatogenic
hydroxyl group on ring B. On the basis of these spectroscopic data,
compound LFQM-113 was elucidated as 13,14-di-toluenesulpho-
nyl-guttiferone-A (6).
The reaction of compound 1 with benzoyl chloride and K2CO3 in
acetone gave compound LFQM-114 (7). The molecular weight of
compound 6 was determined by HRESI-MS to 833.4012 [M+Na]+,
consistent with a molecular formula of C52H58O8Na. The character-
istic monosubstituted aromatic ring signals observed in the 1H
NMR spectra at d 7.35, 7.45 and 8.02 ppm, and the carbon signals
in 13C NMR spectra at d 130.20, 128.49, 133.78 showed indicated
that the two phenolic hydroxyl groups of the substrate were con-
verted to the di-benzoyl ester derivative. Furthermore, this conver-
sion was also supported by the carbonyl peak at 1719 cmÀ1
observed in the IR spectrum.
Shifts in the UV spectrum produced by adding AlCl3 and HCl
indicated the presence of a chelatogenic hydroxyl group. The UV
spectrum displayed absorptions with kmáx (loge) at 339 nm
(1.88), 233 nm (3.76) and 206 nm (3.56), for the pure compound.
Adding AlCl3 produced absorptions at 333 nm (2.52), 232 nm
(3.84) and 207 nm (3.64). Addition of HCl produced absorptions
at 335 nm (2.44), 231 nm (3.87) and 206 nm (3.73) indicating that
the reaction did not occur at the hydroxyl group attached to C-4.
On the basis of these spectroscopic data compound LFQM-114
was identified as 13,14-di-benzoyl-guttiferone-A (7).
Reaction of compound 1 with methanesulfonyl chloride and tri-
ethylamine in CH2Cl2 resulted in compound LFQM-81 (4). The
molecular weight of compound 4 was determined by HRESI-MS
to be 781.3057 [M+Na]+, consistent with a molecular formula of
C
40H54O10S2Na. The 1H NMR signals at d 3.18 and 3.21 were as-
signed as H-39 and H-40, respectively, and the chemical shifts of
C-39 and C-40 at d 32.63, observed in the 13C NMR spectrum, sup-
ported the desired mesylation on positions 13 and 14 of guttifer-
one-A (1). Analysis of IR spectrum of derivative 4 showed two
characteristic bands at 1373 and 1371 cmÀ1, attributed to asym-
metric deformation of the S(@O)2 group, one band at 1180 cmÀ1
,
related to angular symmetric deformation of S(@O)2 and two
bands at 1103.28 and 738.74 cmÀ1 attributed to the axial strain
of the group SOC. The absence of the band related to phenolic hy-
droxyl groups at 3485 cmÀ1 suggested the replacement of two hy-
droxyl groups of the substrate 1. On the basis of these
spectroscopic data, compound LFQM-81 was identified as 13,14-
di-methanesulfonyl-guttiferone-A (4).
Compound LFQM-82 (5) was prepared by reaction of compound
1 with chlorobenzoyl chloride and K2CO3 in acetone. The molecular
weight of compound 5 was determined by HRESI-MS to be
901.3214 [M+Na]+, consistent with
a
molecular formula of
C52H56Cl2O8Na. The chlorobenzoylation of guttiferone-A was sup-
ported by the presence of additional signals of aromatic hydrogen
at d 7.96 (H-41, H-45, H-48 and H-52) and d 7.38 (H-42, H-44, H-49
and H-51), related to the chlorobenzoyl subunit. The presence of
chlorobenzoyl group was also confirmed in the 13C NMR spectrum,
by the signals at d 128.47 attributed to C-42, C-44, C-49 and C-51; d
129.87 attributed to C-41, C-45, C-48 and C-52, d 163.91 (C-39), d
133.77 (C-43), d 124.10 (C-47), d 163.47 (C-46), d 124.48 (C-40),
and d 133.47 (C-50). Based on the absence of bands for axial defor-
mation of OH group in the IR spectrum, it was presumed that all
phenolic hydroxyl groups were substituted. Characteristic bands
were observed at 1749 cmÀ1 and 1253 cmÀ1, corresponding to ax-
ial deformations of C@O and C–O–C@O of the ester group.
2.2. Evaluation of lipophilicity
The lipophilicity of compounds 1–7 were estimated by theoret-
ical calculation of partition coefficient octanol/water (cLogP oct/
wat). The values are shown in Table 1, and clearly indicate that
substituted sulfonyl groups, independent of their volume and size,
contributed to reduce cLogP (oct/wat).

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K. S. T. Dias et al. / Bioorg. Med. Chem. 20 (2012) 2713–2720
2.3. Antimicrobial assay
For Gram-negative microorganisms, all compounds tested
showed a lower activity profile, with the majority of them exhibit-
ing only bacteriostatic properties. Compounds 2 and 7 were active
against P. aeruginosa, while compounds 1, 2 and 5 showed better
fungistatic results against S. typhimurium, in comparison to chlor-
amphenicol used as standard drug. On the other hand, compounds
2, 4, 5 and 7 were the most active against E. coli, showing to be bac-
teriostatic agents, while only compounds 1 and 5 were active
against P. mirabilis, in spite of derivative 5 have showed lower
activity than chloramphenicol.
Differences in the composition of the cell wall of Gram-positive
and Gram-negative bacteria and a possible relationship with the
lipophilic characteristics of the target molecules could be helpful
to explain the difference of these antibacterial compounds.
Gram-positive bacteria possess only one discrete membrane com-
position: acidic polysaccharides (teichoic acids) and negatively
charged phospholipids. In contrast, Gram-negative bacteria pres-
ent two membranes with different chemical constituents: the out-
er membrane containing lipopolysaccharides and the inner
membrane containing negatively charged phospholipids.11
Considering the results obtained for guttiferone-A (1) and its
derivatives 2–7, it is possible to note that compounds 3, 4 and 6
were the most active against Gram-positive bacteria, and that
Guttiferone-A (1) and its derivatives 2–7 were evaluated for
their antimicrobial activities. The results of antimicrobial assays
against a panel of selected Gram-positive, Gram-negative bacteria
and fungi are reported in Table 2 and 3, along with those of refer-
ence drugs chloramphenicol and amphotericin B. DMSO used as
vehicle did not show any effect on bacteria and fungi.
The semisynthetic derivatives 2–7 exhibited a significant action
against Gram-positive S. aureus and B. cereus. For S. aureus, com-
pounds guttiferone-A (1), LFQM-79 (2), LFQM-80 (3), LFQM-81
(4), and LFQM-113 (6) were the most active, with prominent re-
sults for compounds 1, 3, 4 and 6 exhibiting a potency of 1.45-,
3.31-, 3.66- and 4.39-fold, respectively, more active than the stan-
dard drug chloramphenicol. Furthermore, compounds 3, 4 and 6
were about 2.5 times more active than the natural prototype 1,
showing that the modifications carried out on these derivatives
were very important for activity modulation. Against S. epidermidis,
only compound 6 showed activity 3.5-fold greater than that of
chloramphenicol. For B. cereus, only compound 7 was not active,
and all other derivatives were more active than chloramphenicol,
with compounds 3 and 4 showing to be 9.13 and 1.25-fold more
active, respectively, than the natural prototype 1.
Table 2
Antibacterial activity of guttiferone-A (1) and its derivatives 2–7
MO
l
mol/mL
GUT-A (1)
LFQM-79 (2)
LFQM-80 (3)
LFQM-81 (4)
LFQM-82 (5)
LFQM-113 (6)
LFQM-114 (7)
CHL
S. aureus
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
8.293*
18.000*
142.300**
—
0.056*
0.051*
0.177*
—
0.043*
2.743**
2.743*
109.7*
0.171*
1.541*
—
6.034*
8.293**
3.639**
3.293**
12.060**
193.420*
386.840**
4.820*
S. epidermides
B. cereus
—
—
—
—
—
—
—
—
—
—
—
—
0.064*
1.800*
28.400**
58.200*
—
1.647*
0.3545*
123.300*
2.073**
0.227**
1.647**
45.459
—
21.940
—
—
77.360**
48.277*
386.841
15.071*
6.034**
1.507*
**
**
P. aeruginosas
S. typhimurium
E. coli
—
—
—
—
—
—
—
—
—
109.700*
—
—
—
—
—
0.065*
0.057*
—
0.044*
—
49.320*
—
—
—
109.700**
—
0.555*
—
1.818*
—
0.205*
—
0.044*
—
—
—
0.048*
—
—
—
12.060
P. mirabis
165.870*
—
—
—
—
—
—
—
113.649*
—
24.130*
48.270*
—
—, Showed no significant activity.
CHL, Chloramphenicol.
*
Statistically significant difference between CIM50 results. p <0.05.
Statistically significant difference between CIM100 results. p <0.05.
**
Table 3
Antifungal activity of guttiferone-A (1) and its semisynthetic derivatives 2–7
MO
l
mol/mL
GUT-A (1)
LFQM-79 (2)
LFQM-80 (3)
LFQM-81 (4)
LFQM-82 (5)
LFQM-113 (6)
LFQM-114 (7)
FLC
AMB
C. albicans
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
CIM50
CIM100
66.350*
—
—
—
—
—
—
3.639
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
6.530*
—
0.540
—
*
C. krusei
56.900*
142.300**
142.300*
—
52.700*
208.960*
—
—
—
—
—
1.080**
—
C. parapsilosis
C. grabrata
33.175*
—
52.240*
—
—
0.540
—
0.540
—
8.293*
—
56.900*
—
—
7.279*
—
131.700*
208.960*
—
—
—
—
C. tropicalis
33.175*
—
—
—
—
—
3.260*
—
1.080
—
C. neoformans
T. mentagrophytes
M. gypseum
8.293*
16.587
0.555*
1.818**
0.055*
1.818**
1.818*
28.400**
0.056*
3.639**
52.700*
45.459*
43.899*
49.320*
6.530*
**
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1.080**
—
3.293*
52.240*
52.240*
33.175**
165.870*
—
3.639**
—
—
—
—
0.540**
—
7.279**
1.080**
—, Showed no significant activity.
FLC, fluconazole.
AMB, amphotericin B.
*
Statistically significant difference between CIM50 results. p <0.05.
Statistically significant difference between CIM100 results. p <0.05.
**

K. S. T. Dias et al. / Bioorg. Med. Chem. 20 (2012) 2713–2720
2717
Table 4
the phenolic hydroxyl group is a pharmacophoric subunit. For C.
neoformans and dermatophytes T. mentagrophytes and M. gypseum
the prominent derivatives were compounds 2 and 3 that showed
better antifungal activity than the natural benzophenone 1, show-
ing that the insertion of carbonyl functional groups, like an ester
or carbonate, could be auxophoric for the activity. There was not
evidenced any correlation between cLogP (oct/wat) and antifungal
activity among all tested derivatives.
Cytotoxic concentration (CC50) of compounds 1–7
Compound
CC50
(lmol/L)
GUTTIFERONE-A (1)
LFQM-79 (2)
LFQM-80 (3)
LFQM-81 (4)
LFQM-82 (5)
116.00
>142.25
113.55
>131.87
>113.85
>113.33
>123.39
LFQM-113 (6)
LFQM-114 (7)
2.4. Cell viability assay
Cell viability was assessed by MTT reduction assay that is a
rapid and objective assay used to evaluate cellular cytotoxicity,
based on a colorimetric reaction. The values of the cytotoxic con-
centration (CC50) are showed in Table 4 and revealed that all deriv-
atives exhibited higher values of minimum inhibitor concentration
(MIC) required for inhibiting the growth of bacteria and fungi
tested, demonstrating that compound 1 and the derivatives 2–7
are selective and less toxic compounds.
these molecules exhibit the lowest cLogP (oct/wat) value, with
lipophilicity of 9.06, 5.20 and 8.75, respectively. As for Gram-neg-
ative compounds LFQM-79, 82 and 114 that were active against
some Gram-positive bacteria showed higher bactericidal activity
than the other compounds. Despite of the reduced number of ana-
logs, a comparative analysis of the theoretical lipophilicity of anti-
bacterial derivatives was indicative that variation in lipophilicity
could be an important requisite for modulation of the activity
and, as reported in the literature,12 compounds with higher hydro-
philicity are more active against Gram-positive, while a higher
lipophilicity favors the activity against Gram-negative. However,
the correlation between lipophilicity and antimicrobial activity
should be confirmed experimentally and does not exclude the
importance of other structural factors that may be responsible
for the activity of this series of compounds.
3. Experimental section
3.1. General
The 1D NMR spectra were performed on a Varian MR-400 spec-
trometer, operating at 400 MHz for 1H NMR and 100 MHz for 13C
NMR, using CDCl3 as solvent. Chemical shifts are reported in parts
per million (ppm) with reference to tetramethylsilane (TMS). The
coupling constants are reported in hertz (Hz) and signal multiplic-
ities are reported as singlet (s), doublet (d), doublet of doublet (dd),
Among all compounds tested against yeast, only the natural pro-
totype 1 showed significant fungistatic activity in comparison to
Fluconazole and Amphotericin B used as standard drugs. All other
derivatives were not active or showed poor activity, revealing that
Table 5
1H NMR data of guttiferone-A (1) and compounds 2–7 (400 MHz, d ppm, CDCl3)
Position
Compound
GUT-A (1)
LFQM-79 (2)
LFQM-80 (3)
LFQM-81 (4)
LFQM-82 (5)
LFQM-113 (6)
LFQM-114 (7)
7
8
2.51, m, 1H
2.16, m, 2H
2.49, m, 1H
2.15, m, 2H
2.45, m, 1H
2.12, m, 2H
2.20, m, 1H
2.16, m, 2H
2.45, m, 1H
2.17, m, 2H
2.55, m, 1H
2.19, m, 2H
2.45, m, 1H
2.15, m, 2H
12
15
16
7.05, d, 1H, J = 8.5
6.60, d, 1H, J = 2.1
7.44, d, 1H, J = 8.2
7.36, d, 1H J = 1.9
7.39, d, 1H, J = 8.1
7.13, d, 1H, J = 1.9
7.84, d, 1H, J = 8.3
7.44, d, 1H, J = 2.0
7.57, d, 1H, J = 8.4
7.51, d, 1H, J = 3.7
7.50, d, 1H, J = 8.6
7.26, d, 1H, J = 6.0
7.88, d, 1H, J = 7.3
7.59, d, 1H, J = 2.0
6.99, dd, 1H, J = 8.5, 7.41, dd, 1H, J = 8.2, 7.33, dd, 1H, J = 8.1, 7.46, dd, 1H, J = 8.3, 7.95, dd, 1H, J = 8.4, 7.18, dd, 1H, J = 8.6, 7.64, dd, 1H, J = 7.3,
2.1
1.9
1.9
2.0
3.7
6.0
2.0
17
18
19
20
22
23
24
25
27
28
29
30
32
33
34
35
37
38
39
40
41
42
43
44
45
47
48
49
50
51
1.96, m, 2H
1.20, s, 3H
2.61, m, 2H
5.24, m, 1H
1.70, s, 3H
1.48, s, 3H
2.74, m, 2H
5.04, m, 1H
1.57, s, 3H
1.79, s, 3H
2.61, m, 2H
5.15, m,1H
1.53, s, 3H
1.55, s, 3H
2.03, m, 2H
4.92, m, 1H
1.68, s, 3H
1.79, s, 3H
—
—
—
—
—
—
—
—
—
1.90, m, 2H
1.24, s, 3H
2.57, m, 2H
5.20, m, 1H
1.68, s, 3H
1.46, s, 3H
2.64, m, 2H
5.14, m, 1H
1.60, s, 3H
1.68, s, 3H
1.74, m, 2H
5.02, m, 1H
1.52, s, 3H
1.56, s, 3H
2.06, m, 2H
4.85, m, 1H
1.65, s, 3H
1.74, s, 3H
—
1.96, m, 2H
1.07, s, 3H
2.52, m, 2H
5.24, m, 1H
1.59, s, 3H
1.24, s, 3H
2.59, m, 2H
4.97, m, 1H
1.48, s, 3H
1.67, s, 3H
2.48, m, 2H
5.08, m, 1H
1.34, s, 3H
1.41, s, 3H
2.03, m, 2H
4.79, m, 1H
1.50, s, 3H
1.67, s, 3H
—
2.22, s, 6H
—
2.22, s, 6H
—
—
—
—
—
—
—
1.78, m, 2H
1.01, s, 3H
2.46, m, 2H
5.11, m, 1H
1.63, s, 3H
1.45, s, 3H
2.55, m, 2H
4.94, m, 1H
1.60, s, 3H
1.65, s, 3H
2.26, m, 2H
4.96, m, 1H
1.48, s, 3H
1.57, s, 3H
2.04, m, 2H
4.92, m, 1H
1.60, s, 3H
1.65, s, 3H
3.18, s, 6H
3.21, s, 6H
—
1.85, m, 2H
1.26, s, 3H
2.61, m, 2H
5.16, m, 1H
1.67, s, 3H
1.57, s, 3H
2.67, m, 2H
5.04, m, 1H
1.64, s, 3H
1.68, s, 3H
2.54, m, 2H
5.03, m, 1H
1.58, s, 3H
1.60, s, 3H
1.99, m, 2H
4.88, m, 1H
1.64, s, 3H
1.70, s, 3H
—
1.96, m, 2H
1.30, s, 3H
2.59, m, 2H
5.20, m, 1H
1.67, s, 3H
2.03, m, 2H
1.23, s, 3H
2.56, m, 2H
5.16, m, 1H
1.72, s, 3H
1.48, s, 3H
1.46, s, 3H
2.74, m, 2H
5.04, m, 1H
1.61, s, 3H
2.59, m, 2H
5.03, m, 1H
1.67, s, 3H
1.69, s, 3H
1.74, s, 3H
2.59, m, 2H
4.88, m, 1H
1.56, s, 3H
2.52, m, 2H
4.99, m, 1H
1.58, s, 3H
1.58, s, 3H
1.60, s, 3H
2.07, m, 2H
4.77, m, 1H
1.64, s, 3H
1.69, s, 3H
—
7.65, d, 4H, J = 8.1
7.29, d, 4H, J = 7.0
—
7.29, d, 4H, J = 7.0
7.65, d, 4H, J = 8.1
2.45, s, 6H
7.65, d, 4H, J = 8.1
7.29, d, 4H, J = 7.0
—
7.29, d, 4H, J = 7.0
7.65, d, 4H J = 8.1
2.07, m, 2H
4.87, m, 1H
1.67, s, 3H
1.74, s, 3H
—
—
—
—
1.11, s, 9H
1.11, s, 9H
1.11, s, 9H
—
—
—
—
—
—
—
7.99, d, 4H, J = 8.5
7.39, d, 4H, J = 9.3
—
7.39, d, 4H, J = 9.3
7.99, d, 4H, J = 8.5
—
7.99, d, 4H, J = 8.5
7.39, d, 4H, J = 9.3
—
8.02, d, 4H, J = 7.9
7.35, t, 4H, J = 7.8
7.52, t, 2H, J = 7.9
7.35, t, 4H, J = 7.8
8.02, d, 4H, J = 7.9
—
8.02, d, 4H, J = 7.9
7.35, t, 4H, J = 7.8
7.52, t, 2H, J = 7.9
7.35, t, 4H, J = 7.8
—
——
—
—
—
—
—
—
—
—
—
—
—
7.39, d, 4H, J = 9.3

2718
K. S. T. Dias et al. / Bioorg. Med. Chem. 20 (2012) 2713–2720
Table 6
13C NMR data of guttiferone-A (1) and its derivatives 2–7 (100 MHz, d ppm, CDCl3)
Position
Compound
GUTTIFERONE-A (1)
LFQM-79 (2)
LFQM-80 (3)
LFQM-81 (4)
LFQM-82 (5)
LFQM-113 (6)
LFQM-114 (7)
1
2
3
4
5
6
7
8
9
61.87
196.23
114.93
193.83
68.58
50.44
39.02
37.40
206.77
197.74
132.00
115.50
142.78
148.66
113.68
126.98
34.68
17.13
21.72
117.87
134.24
24.73
17.82
30.34
123.01
133.99
24.83
16.83
27.56
118.90
134.11
24.97
18.45
24.41
122.66
133.88
24.66
16.64
—
62.07
193.46
115.93
192.63
68.58
50.64
38.97
37.60
206.78
196.81
131.93
114.92
144.98
149.37
113.76
126.75
34.82
17.13
21.72
119.05
133.99
24.71
17.80
30.18
123.11
133.82
24.81
16.85
28.68
117.93
133.89
24.99
18.55
24.61
122.73
133.77
24.65
16.56
140.76
83.18
26.58
26.58
26.58
—
62.06
193.40
114.91
192.70
68.60
50.63
38.93
37.49
205.70
196.91
133.62
115.82
140.49
144.61
113.93
127.00
34.82
17.13
21.72
117.90
134.00
24.98
17.62
29.98
123.42
133.93
25.02
16.84
27.57
119.31
133.99
25.05
18.54
24.65
122.73
133.84
24.80
16.50
166.45
19.55
166.75
19.72
—
55.53
191.93
114.57
190.14
70.66
51.01
40.06
38.96
205.66
196.18
132.65
115.24
141.09
144.80
113.04
129.0
35.13
18.03
21.51
118.40
136.17
26.88
18.18
30.33
124.04
135.85
26.88
17.54
29.59
118.87
137.71
26.24
18.34
25.64
123.46
135.64
25.82
17.99
32.63
32.63
—
60.36
195.45
115.91
190.27
69.60
51.71
39.96
38.52
207.74
198.03
131.94
116.85
141.89
145.04
113.69
122.29
35.55
18.05
21.00
118.91
135.03
25.70
18.61
29.66
120.15
132.92
25.80
17.85
28.59
118.47
134.77
25.95
19.54
25.45
199.66
132.42
25.63
63.29
194.16
114.82
193.58
69.64
51.67
39.54
38.56
207.37
197.75
132.03
115.99
140.81
144.05
113.34
124.94
35.57
18.09
22.77
118.83
136.60
25.82
18.63
30.59
123.58
133.01
26.02
17.89
28.82
119.94
135.00
26.12
19.62
25.59
122.99
134.87
25.68
55.37
195.71
115.19
190.56
70.71
51.01
39.68
37.71
206.78
197.64
132.00
116.83
142.56
145.50
113.70
125.31
35.20
17.75
22.82
119.30
135.56
24.83
18.18
31.01
123.83
132.09
24.95
17.03
29.73
119.32
135.10
24.98
18.38
24.66
123.29
131.85
24.73
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
17.55
17.64
16.49
163.91
124.48
129.87
128.47
133.77
128.47
129.87
163.47
124.10
129.87
128.47
133.47
128.47
129.87
130.42
128.57
129.84
144.05
129.84
128.57
21.78
127.87
128.57
129.84
145.77
129.84
128.57
21.78
167.11
127.96
130.20
128.49
133.78
128.49
130.20
161.34
127.96
130.20
128.49
134.22
128.49
130.20
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
mulitplet (m). Absorption spectra in the infrared region (IR) were
obtained with a Prestige-21 spectrometer using KBr pellets. Sepa-
rations by column chromatography were carried out with Silica
Gel 60 (60–200 mesh, Merck) and TLC experiments were carried
out on Silica gel 60 F254, supported in aluminum plates (0.2 mm,
Merck). Absorption spectra in the ultraviolet region were collected
with a Shimadzu-2550 dual beam UV–visible spectrophotometer,
as described by Mabry et al.13 with modifications. The phenolic
constituents were dissolved in ethanol (0.1%) and analyzed by
scanning over the range k = 500–200 nm, followed by the addition
of AlCl3 and HCl. All chemicals and solvents were used without
prior treatment, except for anhydrous reactions when solvents
were dried according to the literature.
3.2. General
The fruits of G. brasiliensis were collected from species culti-
vated at University of Viçosa, Minas Gerais, Brazil, where its vou-
cher specimen is deposited under the number VIC2604.
3.3. Extraction and purification of guttiferone-A (1)
The seeds of G. brasiliensis were dried in air, powdered and
extracted by percolation with ethyl acetate at room temperature
for 24 h. The process was repeated until all the material has been
extracted and the solvent was removed under reduced pressure
to furnish a crude ethyl acetate extract (SEAE). SEAE was then

K. S. T. Dias et al. / Bioorg. Med. Chem. 20 (2012) 2713–2720
2719
submitted to column chromatography on silica gel, eluted with
crescent polarity mixtures of hexanes/ethyl acetate to furnish pure
guttiferone-A as a yellow solid. Its structure was confirmed by sev-
eral spectroscopic techniques (IR, UV, MS and NMR) in comparison
to literature data.14
3.4.5. Compound LFQM-113 (6)
0.17 mmol (100 mg) of guttiferone-A (1) was dissolved in 5 mL
of dried CH2Cl2 (5 mL) under N2 atmosphere. After cooling to 5 °C,
0.51 mmol (0.07 mL) of Et3N and 0.51 mmol (65 mg) of TsCl were
added. The reaction was carried out with stirring at room temper-
ature for 8 h, monitored by TLC. When reaction was complete, it
was neutralized with aqueous saturated NaHCO3 and extracted
with ethyl acetate (3 Â 10 mL). The combined organic extracts
were dried over MgSO4, evaporated and purified by preparative
TLC using hexanes (7): EtOAc (3) as eluent, furnishing compound
3.4. Semisynthesis of guttiferone-A derivatives
3.4.1. Compound LFQM-79 (2)
0.17 mmol (100 mg) of guttiferone-A (1) was dissolved in 5 mL
of CH2Cl2. Then 0.34 mmol (0.05 mL) of Et3N, 0.34 mmol (0.08 mL)
of (Boc)2O and a catalytic amount of 4-DMAP were added. The mix-
ture was stirred for a 12 h period at room temperature, until TLC
analysis indicated total conversion. Next, 20 mL of H2O was added
and the reaction mixture was extracted with CH2Cl2 (3 Â 10 mL)
and dried over MgSO4. The combined organic layers were evapo-
rated and the reaction product was purified by column chromatog-
raphy on silica gel using hexanes (9): ethyl acetate (1) as eluent.
Compound 2 was obtained in 87% yield. IR spectrum mmax CH2Cl2
cmÀ1: 3446, 2981, 2931 (C–H), 1766, 1647, 1579, 1257. HRMS
Calcd for C43H58O8 [M+Na]+, 725.4023, Found 725.4033. 1H and
13C NMR data are presented in Tables 5 and 6.
6 as a pale yellow oil in 65% yield. IR spectrum mmax CH2Cl2 cmÀ1
2960, 1668, 1478, 1433, 1260, 1184, 1118, 1021, 799, 749, 716,
540. HRMS Calcd for C52H62O10S2 [M+Na]+, 933.3676, Found
933.3645. 1H and 13C NMR data are presented in Tables 5 and 6.
:
3.4.6. Compound LFQM-114 (7)
0.17 mmol (100 mg) of guttiferone-A (1) was dissolved in 5 mL
of dried CH2Cl2. Then, 0.34 mmol (47.00 mg) of K2CO3 and
0.34 mmol (0.04 mL) of BzCl were added in sequence. The reaction
mixture was stirred at room temperature for a 12 h period. After
total conversion of the starting material observed by TLC, two por-
tions of 30 mL of CH2Cl2 were added and the resultant solution
was washed with aqueous saturated NaHCO3. The organic extract
was dried over MgSO4, evaporated and purified by preparative TLC
using hexanes (9.5): EtOAc (0.5) as eluent, affording compound 7
in 51% yield. IR spectrum mmax CH2Cl2 cmÀ1: 2964, 2916, 1749,
1600, 1681, 1246. HRMS Calcd for C52H58O8 [M+Na]+, 833.4023,
Found 833.4012. 1H and 13C NMR data are presented in Tables 5
and 6.
3.4.2. Compound LFQM-80 (3)
To a solution of 0.17 mmol (100 mg) guttiferone-A (1) in CH2Cl2
(5 mL), were added 1 mL (10.5 mmol) of acetic anhydride and cat-
alytic amount of 4-DMAP. The system was kept under stirring at
room temperature for 4 h, monitored by TLC. After reaction was
complete, water were added (20 mL) to the mixture, the aqueous
phase was extracted with ethyl acetate (3 Â 20 mL) and dried with
sodium sulfate. The combined organic layers were concentrated
and the crude product was purified by column chromatography
on silica gel using hexanes (9): ethyl acetate (2) as eluent. Pure
compound 3 was obtained in 81% yield. IR spectrum mmax CH2Cl2
cmÀ1: 2970, 2922, 1778, 1666, 1608, 1201. HRMS Calcd for
3.5. The evaluation of lipophilicity by cLogP (oct/water)
Lipophilicity values were estimated through determination the-
oretical of cLogP (oct/wat) by using the QikProp program.15 The
QikProp program calculates the cLogP (oct/wat) values from
regression equations using experimental data and molecule phys-
ical descriptors (hydrogen bond counts, atom types and charges,
rotor counts, etc.) through Monte Carlo statistical mechanics sim-
ulations.16 Calculated lipophilicity expressed by cLogP (oct/wat)
of compounds 1–7 are showed on Table 1.
C
42H54O8 [M+Na]+, 709.3710, Found 709.3709. 1H and 13C NMR
data are presented in Tables 5 and 6.
3.4.3. Compound LFQM-81 (4)
0.17 mmol (100 mg) of guttiferone-A (1) was dissolved in dried
CH2Cl2 (5 mL) under N2 atmosphere. After cooling to 5 °C,
0.34 mmol (0.05 mL) of Et3N and 0.34 mmol (0.03) of MsCl were
added. The reaction was carried out with stirring, at room temper-
ature for a 8 h period. After reaction was complete (TLC), water
were added (20 mL), the aqueous phase was extracted with ethyl
acetate (3 Â 20 mL), dried with sodium sulfate and concentrated
under reduced pressure, affording the mesylate 4 in 55% yield.
IR spectrum mmax CH2Cl2 cmÀ1: 2968, 2929, 1732, 1662, 1575,
1180, 1103. HRMS Calcd for C40H54O10S2 [M+Na]+, 781.3050,
Found 781.3057. 1H and 13C NMR data are presented in Tables 5
and 6.
3.6. Antimicrobial activity evaluation
Guttiferone-A (1) and its derivatives 2–7 were evaluated for
their antimicrobial activities against the fungi through a standard
RPMI medium with L-glutamine and morpholinepropanesulfonic
acid buffer broth microdilution method proposed by document
M27A3 (CLSI, 2008)17 and supplemented with 2% dextrose to
yeasts and document M38A2 (CLSI, 2003)18 to filamentous fungi
and a standard Mueller Hinton broth microdilution method for
bacteria proposed by document M7A6 (CLSI, 2003).19 The stock
solutions of compounds 1–7 were prepared in DMSO 1% at final
3.4.4. Compound LFQM-82 (5)
concentration and tested at concentrations (lg/mL) 100; 62.5;
Guttiferone-A (1) (0.17 mmol) was dissolved in 5 mL acetone.
Then, 0.34 mmol (47 mg) of K2CO3 and 0.34 mmol (0.04 mL) of 4-
ClBzCl were added in sequence. The reaction mixture was stirred
at room temperature for a period of 4 h. After total conversion of
the starting material (TLC), the reaction material was neutralized
with aqueous saturated NaHCO3 and extracted with ethyl acetate
(3 Â 10 mL). The combined organic extracts were dried over
MgSO4, evaporated and purified by preparative TLC using hexanes
(9): EtOAc (1) as eluent, furnishing compound 5 as a pale yellow oil
in 60% yield. IR spectrum mmax CH2Cl2 cmÀ1: 2976, 2926, 1770,
31.2; 15.6; 7.8; 3.9; 1.95; 0.48; 0.24; 0.06. The standard drug chlor-
amphenicol was applied as a control of antibacterial action at con-
centrations (
Amphotericin B as a control of fungicidal action at concentrations
g/mL) 8; 4; 2; 1; 0.5; 0.25; 0.12; 0.06; 0.03; 0.015; and Fluconaz-
ole as a control of fungistatic action at concentration ( g/mL) 64;
lg/mL) 8; 4; 2; 1; 0.5; 0.25; 0.12; 0.06; 0.03; 0.015;
(l
l
32; 16; 8; 4; 2; 1; 0.5; 0.25; 0.125; 0.0625; 0.03125. The micro-
plates were incubated at 35 °C for 24 h for bacterial, 37 °C for
24 h for yeast and 30 °C for 48 h for filamentous fungi. Results were
visualized and analyzed by spectrophotometry. The inhibitory con-
centration of microbial growth was determined at 50% (IC50) and
1668, 1652, 1252. HRMS Calcd for
C
52H56Cl2O8 [M+Na]+,
901.3244, Found 901.3214. 1H and 13C NMR data are presented
in Tables 5 and 6.
100% (IC100) in
lmol/mL and compared for each compound and
microorganism. The tests were all done in duplicates.

2720
K. S. T. Dias et al. / Bioorg. Med. Chem. 20 (2012) 2713–2720
3.7. Cell viability assay
of semisynthetic derivatives could be explored as new drug proto-
type candidates to antimicrobial agents with easy access from an
abundant natural product, with low toxicity and good selectivity.
Further studies should be conducted to better understand the
mechanism of action of these novel molecules and their effective-
ness in in vivo models.
Cell viability was determined using a modified MTT assay with
peripheral human blood mononuclear cells (PBMC) obtained from
healthy volunteers by Ficoll-Hypaque density gradient centrifuga-
tion. The cell suspension of normal PBMC at a concentration of
2.4 Â 106 cells/mL was distributed in a 96-well plate, 90
lL in each
well with 10
lL of test compounds at different concentrations,
Acknowledgments
incubated at 37 °C in an incubator at 5% CO2. In this assay, concen-
trations used were 100, 62.5, 31.2, 15.6, 7.8, 3.9, 1.95, 0.48, 0.24,
0.06 lg/mL of the compounds tested and the plate was incubated
for 48 h. After the incubation period, the morphology of cells of
control and test wells were observed microscopically. After, it
This work was supported by grants from INCT de Fármacos e
Medicamentos (INOFAR, CNPq, Brazil, #573.564/2008-6), Universal
Project (CNPq, Brazil, #471178/2007-1) and Universal Project (Fap-
emig, Brazil, #CEX-APQ-01072-08). The authors are also grateful
for the fellowships granted by CAPES, CNPq, FAPEMIG and to Pro-
fessor Norberto P. Lopes and José Carlos Tomás for HRESI-MS
analysis.
was added 10
incubated again for an additional 4 h period. Then, the medium
was carefully removed and added to 100 L of DMSO for solubili-
lL of the dye MTT (5 mg/mL) and the cells were
l
zation of formazan crystals. The plates were shaken for 5 min
and absorbance for each sample was measured in a spectrophoto-
metric microplate reader at 560 nm. The absorbance obtained from
control cells, untreated, was taken as 100% cellular viability.20 Data
were analyzed using linear regression to obtain values for CC50
(cytotoxic concentration for 50% of cells).
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