Guanidinium compounds with sub-micromolar activities against Mycobacterium tuberculosis. Synthesis, characterization and biological evaluations

Article abstract of DOI:10.1016/j.bmc.2015.07.053

Seven polycharged species, incorporating 1, 2, 3, 4 and 6 guanidine arms organized around a benzene core were synthesized and assayed as anti-mycobacterial agents against Mycobacterium tuberculosis. They display MIC values comprised between 25 and 12.5 μM (close to ethambutol EMB) for the mono- and the hexa-substituted derivatives, and 0.8 μM (close to isoniazid and streptomycin) for the tri-substituted derivative. The three bi- and the tetra-substituted analogs displayed MIC values of ca. 6.5-3.0 μM. The latter were also evaluated against the isoniazid-resistant MYC5165 strain, resulting in highly interesting micromolar or sub-micromolar MIC, ca. 4-125 times more active than isoniazid. These preliminary results are attractive for the development of new anti-TB agents.

Full text of DOI:10.1016/j.bmc.2015.07.053

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Guanidinium compounds with sub-micromolar activities against
Mycobacterium tuberculosis. Synthesis, characterization and
biological evaluations
Hugues Massimba-Dibama a, , Maxime Mourer a,b, Patricia Constant c,d, Mamadou Daffé c,d
,
Jean-Bernard Regnouf-de-Vains a,b,
⇑
a CNRS, SRSMC, UMR 7565, équipe MoBAT, F-54506, France
b Université de Lorraine, SRSMC, UMR 7565, équipe MoBAT, Vandœuvre-lès-Nancy F-54506, France
c CNRS, IPBS (Institut de Pharmacologie et Biologie Structurale), Département Mécanismes Moléculaires des Infections Mycobactériennes, 205 route de Narbonne, F-31077
Toulouse Cedex 04, France
d Université de Toulouse, UPS, 118 route de Narbonne, F-31062 Toulouse Cedex 9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven polycharged species, incorporating 1, 2, 3, 4 and 6 guanidine arms organized around a benzene core
Received 29 May 2015
Revised 17 July 2015
Accepted 25 July 2015
Available online xxxx
were synthesized and assayed as anti-mycobacterial agents against Mycobacterium tuberculosis. They dis-
play MIC values comprised between 25 and 12.5
hexa-substituted derivatives, and 0.8 M (close to isoniazid and streptomycin) for the tri-substituted
derivative. The three bi- and the tetra-substituted analogs displayed MIC values of ca. 6.5–3.0 M. The
lM (close to ethambutol EMB) for the mono- and the
l
l
latter were also evaluated against the isoniazid-resistant MYC5165 strain, resulting in highly interesting
micromolar or sub-micromolar MIC, ca. 4–125 times more active than isoniazid. These preliminary
results are attractive for the development of new anti-TB agents.
Keywords:
Ammonium
Guanidinium
Tuberculosis
Ó 2015 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis
Anti-mycobacterial
1. Introduction
nitroimidazole-like PA-824, the 1,2-diamine SQ-109, the rifamycin
derivative rifapentine and the diarylquinoline bedaquiline, the first
Data from WHO concerning tuberculosis (TB), lead to an estima-
tion of 9 million new cases in 2013 (6.1 million declared), with
1.5 million deaths (0.36 million HIV-positive people). Overall,
emergence of multidrug-resistant tuberculosis resulted in an esti-
mated population of 0.48 million people, the double of 2011’ esti-
mation, with 0.17 million deaths.1 This airborne and contagious
infectious disease that is preventable and curable is the second
cause, after HIV, of human death due to a single etiologic agent,
Mycobacterium tuberculosis. Approaches to fight TB are related to
hygienic strategies, to detection, to vaccination and to drug discov-
ery.2 More particularly, a new diagnostic to detect rapidly M. tuber-
culosis sensitive or resistant to rifampicin has been launched very
recently.
novel TB drug approved in 2012, since ca. 40 years. In Phase III, are
the nitroimidazole-like delamamid, the rifapentine and the fluoro-
quinolones gatifloxacin and moxifloxacin.3–8
Engaged in a research programme devoted to the discovery of
new antibacterial agents targeting the bacterial membrane denat-
uration through expected supramolecular interactions between
highly organized cations and anionic membrane constituents, we
have recently shown that pre-organizing guanidinoethyl groups
at the upper rim of calix[4]arene was a powerful way to generate
an antibacterial activity while the constitutive phenolic monomer
is inactive.9 Such genesis of biological activity lead us to engage a
wider programme devoted to evaluation of the antibacterial activ-
ity of the resulting tetra-para-guanidinoethylcalix[4]arene (CX1)
against reference and resistant bacterial strains,10–13 and develop-
ment of physico-chemical investigations at the level of bacterial
and model membrane.14–17
Some very recent reviews have focused on the past, present and
future of anti-tuberculosis drugs. Currently, seven drugs are in
Phase II, the oxazolidinones AZD5847, linezolid and sutezolid, the
In parallel to Gram positive and Gram negative bacteria, we
recently found that CX1 and some of its derivatives were very
interestingly active against reference and INH-resistant M. tubercu-
losis strains.18
⇑
Corresponding author. Tel.: +33 3 83684324.
E-mail address: jean-bernard.regnouf@univ-lorraine.fr (J.-B. Regnouf-de-Vains).
Present address: Département des Sciences Physiques, Ecole Normale Supérieure,
Libreville, Gabon.
http://dx.doi.org/10.1016/j.bmc.2015.07.053
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Massimba-Dibama, H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.053

2
H. Massimba-Dibama et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 1. From 3D-constrained CX1 to 2D-constrained 30.
For example, CX1 showed Minimum Inhibitory Concentration
(MIC) values at same low level of 1 M) for both
g mLꢀ1 (0.8
37Rv reference and INH-resistant MYC5165 M. tuberculosis
strains.
trifluoroacetic acid (TFA) in CH2Cl2, at rt, to afford, after evapora-
l
l
tion of solvent and TFA, then multiple trituration/filtration
sequences carried out in Et2O, the corresponding ammonium salts
3, 8, 13, 18, 23, 28 and 33 respectively, in 96%, 71%, 92%, 85%, 98%,
80%, and 88% yields.
H
These results opened an interesting area of investigations,
focusing on the nature of the organizing core. In this sense, as
the previous active calixarenic guanidinium derivative displays a
high 3D-constraint level, organizing the guanidinium arms as a tet-
rapod, we propose here to diminish this constraint level, through a
higher degree of freedom given by a simple benzene ring. For this
purpose, the synthesis of a flattened tetraguanidinium analog of
CX1 (Fig. 1), was obviously engaged. In the same time, in order
to define preliminary structure activity relationships, other
2D-constrained benzene derivatives including from 1 to 6 guani-
dinium arms were prepared and evaluated, object of the present
work.
The 2D-constrained systems that were prepared are shown in
Scheme 1. The molecules are, as the seminal calixarene, conceived
as poly-guanidine compounds, in which the guanidine functions
are para-guanidinoethyl-phenoxy-methylene groups attached to
the central phenyl core across its tolyl, xylyl, mesityl, durenyl
and mellitenyl derivatives.
The nucleophilic attack of the free amines issued from these
salts onto N1,N2-(di-Boc)-N3-triflylguanidine
B afforded the
(di-Boc)-guanidino derivatives 4, 14, 19, 24, 29 and 34, with yields
of 85%, 98%, 90%, 87%, 61% and 84%, respectively. As an example, 9
was also prepared from 7, 1,3-bis-(tert-butoxycarbonyl)- 2-methy
l-2-thiopseudourea and HgCl2 in dry DMF with a yield of 94%.
The expected guanidiniums 5, 10, 15, 20, 25 and 35 were finally
prepared by a treatment similar to previous ammonium salts 3–33,
and a final lyophilization from distilled water solutions. The guani-
dinium trifluoroacetate salts were obtained with a yield of 94%,
75%, 98%, 79%, 90%, 71%, and 79%, respectively.
All compounds were fully characterized, notably by 1H and 13C
NMR, mass spectrometry and elemental analyses. For the final
guanidinium derivatives, elemental analyses were consistent with
the expected salt structures, accompanied by some molecules of
H2O resulting from the lyophilization process.
The compounds tested in this study are the mono-substituted
(tolyl) species 5, the 1,2-, 1,3- and 1,4-di-substituted (xylyl) species
10, 15 and 20, the 1,3,5-tri-substituted (mesityl) species 25, the
1,2,4,5-tetra-substituted (durenyl) species 30 and the
1,2,3,4,5,6-hexa-substituted (mellitenyl) species 35. All these com-
pounds show interesting antibacterial activities against various
Gram positive and Gram negatives bacterial strains, associated to
low-to modest cytotoxicities.19
2.2. Biological evaluations
In this study, in vitro antimycobacterial activity (e.g., Minimum
Inhibitory Concentration (MIC) and 50% Inhibitory Concentration
(IC50) determination) of compounds 5, 10, 15, 20, 25, 30 and 35
was evaluated against the reference strain M. tuberculosis H37Rv
and the INH-resistant strain MYC5165 (M. tuberculosis strain
mutated in InhA).21,22 The results of these evaluations are con-
densed in Table 1. MIC and IC50 values are given in molar and mass
dimensions.
2. Results and discussion
2.1. Chemistry
The compound 5, designed as monomeric species for compar-
ison with poly-guanidinium species, is the less active of the series,
with a MIC of 25
Similar results were obtained for the hexa-substituted species 35.
Even close to ethambutol (EMB, 9.8 M), 5 and 35 were not evalu-
ated against the INH-resistant strain. The ortho-di-substituted spe-
lM (9.8 lg/mL) and an IC50 of 5.9 lM (2.3 lg/mL).
The general synthetic pathway leading to final guanidylated
compounds 5, 10, 15, 20, 25, 30 and 35 involved fours steps from
the starting bromomethylated phenyl species 1, 6, 11, 16, 21, 26
and 31 (Scheme 2). The latter were commercially available, but
the tri-substituted species 21 was synthesised according to a pro-
cedure adapted from Li et al.20 by radical bromination of mesity-
lene with NBS in CCl4.
The bromides were reacted with ca. stoichiometric amounts of
para-[(Boc)aminoethyl]phenol A (Boc-tyramine) in refluxing ace-
tonitrile, in the presence of K2CO3 as base. The corresponding
Boc-amino derivatives 2, 7, 12, 17, 22, 27 and 32 were obtained
with yields of 90%, 62%, 93%, 97%, 70%, 98% and 98%, respectively,
after chromatography. The latter were then treated with
l
cies 10 exhibits MIC value of 6.2
(1.0
select it for evaluation against the MYC5165 strain.
Nevertheless, 10 showed moderate activity of 3.1
(2.2 g/mL) against this strain, close to ciprofloxacin (2.5
l
M (4.2
l
g/mL) and IC50 of 1.5
l
M
lg/mL), also close to EMB, but at a level we found justified to
a
lM
l
l
M),
and 4 times as active as INH. The meta- (15) and para- (20)
di-substituted species were judged identical in terms of MIC and
IC50 against H37Rv strain with MIC values of 3.1
close to ciprofloxacin, and IC50 around 0.8 M (ca. 0.6
compounds exhibit a submicromolar activity (0.8 M) against the
lM (ca. 2
lg/mL)
l
l
g/mL); both
l
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H. Massimba-Dibama et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
Scheme 1. Compounds of the study.
MYC5165 strain, similar to CX1 calixarene species,18 and 16 times
as active as INH.
superior to that of the latter. Such differences should result, if we
consider that 30 keeps this flat conformation, in strong differences
of activity. The biological results given in Table 1 show that 30 is
three-times less active than CX1 against H37Rv, but, very interest-
ingly, is ca. eight times more active against MYC5165. This differ-
ence of activity, to the benefit of compound 30, is reinforced by
the selectivity indices. Compound 30 appears in fact more cyto-
The symmetric tri-substituted derivative 25 appears very active
against both strains, with H37Rv MIC value of 0.8
close to INH and streptomycin and a four-fold lower MIC against
MYC5165 (0.2 M; 0.2 g/mL).
The tetra-substituted derivative 30 exhibits a modest activity
against H37Rv strain at 3.2 M (4.3 g/mL), but appears ca. 16
times more active against the INH-resistant MYC5165 strain, with
a sub-micromolar MIC (0.1 M; 0.13 g/mL), making it the leader
lM (0.8 lg/mL),
l
l
l
l
toxic than CX1, making it less interesting for H37Rv strain
(SI30 = 11.5 vs SICX1 >256), but much more pertinent when evalu-
ated against the INH resistant one, with a SI30 around 380 (vs
SICX1 >256). These results show that it is possible to maintain a
high anti-mycobacterial activity by keeping close together four
guanidinium ions, whatever nature of the organizing platform, cal-
ixarenic or benzenic.
l
l
of this family. Overall, 30 appears eight times more active than
its tetra-guanidino-calixarene analog CX1.18 The compounds
bis-meta 15, bis-para 20, tris 25 and tetra 30 are active at
sub-micromolar concentrations against MYC5165 strain, that
makes them, respectively, 16, 60 and more than 125 times more
active than INH against this strain.
3. Conclusion
These results were compared to cellular cytotoxicity values pre-
viously obtained for these compounds on non-cancerous human
pulmonary embryonic fibroblasts MRC5 cells,19 in order to deter-
mine the corresponding Selectivity Indices (SI). The results given
in Table 2 show that, with regards to the H37Rv strain, the SI are
relatively low, comprised between 1 and 50, the higher value being
obtained with the tri-substituted derivative 25, which displays a
sub-micromolar activity but a modest cytotoxicity (SI = 49).
With regards to the INH-resistant strain MYC5165, the Table 2
reveals that the SI become highly advantageous for two species,
25 (SI: 198) and, above all, the tetra-substituted derivative 30
which acquires the leader character of this series, with a SI around
380.
As mentioned at the beginning of this study, the
tetra-substituted analog 30 has been designed to feature a flat-
tened analogy of the 3D-tetra-para-guanidinocalix[4]arene CX1.
The representation of 30 in flat conformation and CX1 in conical
conformation (Fig. 2) shows that 30 is approximately twice as wide
as CX1, corresponding to a surface occupancy four to five times
In this investigation, seven guanidinium-containing compounds
based on a benzene organizing platform and displaying between
one and six arms, have been prepared and characterized, then sub-
jected to antibacterial evaluation against INH-sensitive and resis-
tant M. tuberculosis strains. Except the mono- and the
hexa-substituted derivatives, all compounds displayed Minimum
Inhibitory Concentrations close to
1 lM, notably the
tri-substituted derivative which exhibits a sub-micromolar MIC
against the INH-sensitive strain H37Rv. More particularly, four of
these compounds, the bis-meta-, the bis-para, the tri- and the
tetra-substituted species 15, 20, 25 and 30, respectively, showed
a sub-micromolar activity accompanied for 15, 25 and 30 by a high
selectivity index of ca. 62, 200 and 380, respectively when tested
on the INH resistant MYC5165 strain. In the present case, the fact
that a positive discrimination between the standard H37Rv and
the INH-resistant MYC5165 M. tuberculosis strains is observed for
the compounds 15, 25 and 30 calls for deeper investigations within
this new family of polycationic substances. These results make this
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4
H. Massimba-Dibama et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Scheme 2. Reagents and conditions: (i) A, K2CO3, MeCN, reflux, TLC monitoring; (ii) CH2Cl2, TFA, 85:15, rt, TLC monitoring; (iii) B, NEt3, CH2Cl2, MeOH or C, HgCl2, DMF, TLC
monitoring; (iv) CH2Cl2, TFA, 85:15, rt, TLC monitoring.
Table 1
Table 2
Anti-mycobacterial activities of guanidinium derivatives 5, 10, 15, 20, 25, 30 and 35
against reference H37Rv and INH-resistant MYC5165 M. tuberculosis strains
Selectivity indices calculated (CC50 24 h19/MIC) for the reference H37Rv and INH-
resistant MYC5165 M. tuberculosis strains, after 24 h exposure of MRC-5 cells to the
compounds of the study
Compounds
(MW)
H37Rv
MYC5165
M) IC50
g/mL) g/mL)
H37Rv
SI
MYC5165
SI
MIC (
l
M)
IC50
(
l
M)
MIC (
l
(lM)
(lg/mL)
(lg/mL)
(l
(l
5
10
15
20
25
30
26.0:9.8
100.0:4.2
34.5:2.2
21:2.13
39.5:0.8
49:4.3
2.6
24
16
10
49
ND
100:2.2
34.5:0.56
21:0.55
39.5:0.2
49:0.13
ND
45
62
38
198
377
5
25
(9.8)
6.2
(4.2)
3.1
5.9
(2.3)
1.5
(1.0)
0.7
ND
ND
(392.37)
10
(706.60)
15
3.1
0.9
(2.2)
0.8
(0.63)
0.3
11.5
1
35
26.0:24.4
>256:1
>10:0.08
>10:2
(697.6)
20
(688.62)
25
(1002.88)
30
(1326.15)
35
(2.2)
3.1
(2.13)
0.8
(0.8)
3.2
(4.3)
12.5
(24.4)
(0.5)
0.8
(0.55)
0.35
(0.35)
0.5
(0.7)
3.8
(7.4)
(0.56)
0.8
(0.55)
0.2
(0.2)
0.1
(0.13)
ND
(0.21)
0.2
(0.14)
ND
(ND)
0.03
(0.04)
ND
CX1
INHa
EMBa
>256
>125
>5
>256:1
>10:1.7
>256
>6
Concentrations in
lg/mL; ND: not determined.
a
From Ref. 23.
(1954.60)
(ND)
(ND)
4. Experimental section
4.1. Chemistry
Reference compounds
CX1 0.8
(1239.00)a
INH
(137.14)
EMB
(277.15)
Ciprofloxacin
(331.40)
Streptomycin
(581.57)
0.25
(0.31)
0.22
(0.03)
ND
0.8
(1)
12.5
(1.7)
0.1
(1)
0.6
(0.08)
9.8
(2)
2.5
(0.8)
0.7
(0.12)
0.65
(0.9)
Melting points (°C, uncorrected) were determined on an
Electrothermal 9200 in Capillary apparatus. 1H and 13C spectra
were recorded on a Bruker DRX 400 (chemical shifts in ppm).
Mass spectra (electrospray—ES) were recorded on a Bruker
Daltonics micrOTOF-Q apparatus, at the Service Commun de
Spectrométrie de Masse Organique, Nancy. Infrared measurements
0.6
(0.2)
2.5
(0.8)
2.7
1.8
(0.6)
(0.4)
(1.6)
were performed on a Vector 22 Bruker FT apparatus (KBr,
cmꢀ1) and UV spectra were recorded on a SAFAS UV mc2 apparatus
(kmax in nm,
in dm3 molꢀ1 cmꢀ1. Elemental analyses were per-
c in
ND: not determined.
a
See Ref. 18.
e
formed at the Service de Microanalyse, Nancy. Merck TLC plates
were used for chromatography analysis (SiO2, ref 1.05554; Al2O3,
ref 1.05581). All commercially available products were used with-
out further purification unless otherwise specified.
family highly attractive for deepening investigations related to the
mode of action notably with regards to the bis-guanidinium strep-
tomycin, their synergistic behavior and in vivo assays.
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H. Massimba-Dibama et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 2. Chem3D side-representation of 30 and CX1 (not minimized).
4.1.1. Compounds 2, 7, 12, 17, 22, 27, 32—general procedure
A mixture of N-Boc-tyramine A and K2CO3 in dry MeCN was
refluxed under Ar during 30 min. The bromomethylbenzene
derivative was added and reflux was continued during 24 h (TLC
monitoring; SiO2, CH2Cl2/MeOH 99.8:0.2 to 98:2). After cooling,
the solvent was evaporated to dryness, and the solid residue was
dissolved in CH2Cl2. The insoluble materials were filtered off, and
the concentrated filtrate was chromatographed (SiO2;
CH2Cl2/MeOH X:Y%) to give the desired n-[para-(Boc-
aminoethyl)-phenoxymethyl] benzene.
1.52 10ꢀ3 mol), dry MeCN (25 mL) and m-dibromomethylbenzene
11 (0.20 g, 0.76 10ꢀ3 mol) (TLC monitoring; SiO2, CH2Cl2/MeOH
98:1). Chromatography (SiO2; CH2Cl2/MeOH 100:0 to 98:2). 12
(0.41 g, 0.71 10ꢀ3 mol, 93.0%). White powder. Mp: 132–134 °C. IR
(KBr): 1683.35 (CO). UV–vis (CH2Cl2): 277.0 (3324). 1H NMR
(400 MHz, CDCl3): 1.43 (s, 18H, Me3C); 2.73 (t, J = 6.92 Hz, 4H,
CH2CH2NH); 3.34 (br s, 4H, CH2CH2NH); 4.52 (br s, 2H, NH); 5.06
(s, 4H, ArOCH2); 6.91 (d, J = 8.84 Hz, 4H, ArH); 7.10 (d, J = 8.56 Hz,
4H, ArH); 7.39 (br s, 3H, ArH); 7.50 (s, 1 H, ArH). 13C NMR
(100 MHz, CDCl3): 28.4, 35.4, 42.3, 69.9, 79.4, 115.0, 126.5, 127.0,
128.9, 129.8, 131.4, 137.5, 155.9, 157.4. Anal. calcd for
4.1.1.1.
(2).
a
-[para-(Boc-aminoethyl)-phenoxy]toluene
(0.10 g, 0.42 10ꢀ3 mol), K2CO3 (0.06 g,
L,
C34H44O6N2 (576.32): C, 70.81; H, 7.69; N, 4.86; found: C, 70.52;
From
A
H, 7.79; N, 4.88. ES-MS (pos. mode): 577.23 [M+H]+, 599.25
[M+Na]+, 615.24 [M+K]+.
0.42 10ꢀ3 mol), dry MeCN (15 mL), benzyl bromide 1 (50
l
0.42 10ꢀ3 mol) (TLC monitoring; SiO2, CH2Cl2/MeOH 2%).
Chromatography: SiO2; CH2Cl2/MeOH 100:0 to 98:2. 2 (0.13 g,
90%). White solid. Mp: 81–83 °C. IR (KBr): 3360.66 (NH); 1681.61
(CO). UV–vis (CHCl3): 277 (2960.3). 1H NMR (400 MHz, CDCl3):
1.46 (s, 9H, Me3C); 2.74 (t, J = 7.04 Hz, 2H, ArCH2CH2NH); 3.34 (br
s, 2H, ArCH2CH2NH); 4.62 (br s, 1 H, NH); 5.05 (s, 2H, ArOCH2);
6.93 (d, J = 8.56 Hz, 2H, ArH); 7.12 (d, J = 8.56 Hz, 2H, ArH); 7.33–
7.44 (m, 5H, ArH). 13C NMR (100 MHz, CDCl3): 28.53; 35.60;
42.21; 70.70; 79.52; 114.20; 127.21; 128,32; 128.80; 129.00;
4.1.1.4.
(17).
Bis-[
From
a
,
a0-[para-(Boc-aminoethyl)-phenoxy]]-p-xylene
(1.13 g, 4.76 10ꢀ3 mol), K2CO3 (0.63 g,
A
4.54 10ꢀ3 mol), dry MeCN (40 mL), and p-dibromomethylbenzene
16 (0.6 g, 2.27 10ꢀ3 mol) (TLC monitoring SiO2, CH2Cl2/MeOH
98:2). Chromatography (SiO2, CH2Cl2/MeOH 100:0 to 98:2). 17
(1.28 g, 97%). White solid. Mp: 173–174 °C. IR (KBr): 3360.55
(NH); 1681.84 (CO). UV–vis (CH2Cl2): 276 (4600) 1H NMR
(400 MHz, DMSO-d6): 1.36 (s, 18H, Me3C), 2.61 (t, J = 7.1 Hz, 4H,
CH2CH2NH), 3.08 (m, 4H, CH2CH2NH), 5.07 (s, 4H, CH2Ph), 6.84
(m, 2H, NH), 6.92 (d, J = 8.2 Hz, 4H, ArH), 7.09 (d, J = 8.2 H, 4H,
ArH), 7.44 (s, 4H, ArH). 13C NMR (100 MHz, CDCl3): 28.83, 35.71,
42.36, 70.16, 79.59, 115.34, 128.09, 130.18, 131.77, 137.26,
156.30, 157.78. Anal. calcd for C34H34O6N2 (576.72): C, 70.81; H,
7.69; N, 4.86; found: C, 70.51; H, 7.66; N, 4.92. ES-MS (pos. mode):
599.2 [M+Na+]+.
131.80; 141.20; 151.02; 157.90. Anal. calcd for
C20H25NO3
(327.42): C, 73.37; H, 7.70; N, 4.28; found: C, 73.45; H, 7.64; N,
4.34.
ES-MS
(pos.
mode):
350.20
[M+Na]+,
228.05
[MꢀCOOCMe3+2H]+, 211.02 [MꢀCOOCMe3ꢀNH]+.
4.1.1.2.
(7).
Bis-[
From
a,
a0-[para-(Boc-aminoethyl)-phenoxy]]-o-xylene
A
(0.36 g, 1.52 10ꢀ3 mol), K2CO3 (0.21 g,
1.52 10ꢀ3 mol), dry MeCN (20 mL) and o-dibromomethylbenzene
6 (0.20 g, 0.76 10ꢀ3 mol). (TLC monitoring; SiO2, CH2Cl2/MeOH
98:2). Chromatography (SiO2; CH2Cl2/MeOH 100:0 to 98:2). 7
(0.27 g, 62%). White powder. Mp: 110–112 °C. IR (KBr): 3375.0 (–
CONH–); 1685.1 (CO). UV–vis (CHCl3): 276 (3067.0). 1H NMR
(400 MHz, CDCl3): 1.43 (s, 18H, Me3C); 2.73 (t, J = 6.8 Hz, 4H,
CH2CH2NH); 3.33 (br t, J = 8.0 Hz, 4H, CH2CH2NH); 4.53 (br s, 2H,
NH); 5.15 (s, 4H, ArOCH2); 6.90 (d, J = 8.56 Hz, 4H, ArH); 7.09 (d,
J = 8.28 Hz, 4H, ArH); 7.36 (m, 2H, ArH); 7.50 (m, 2H, ArH). 13C
NMR (100 MHz, CDCl3): 28.42, 35.39, 42.01, 68.05, 79.38, 114.95,
128.43, 128.94, 129.75, 131.50, 135.20, 155.97, 157.30. Anal. calcd
for C34H44O6N2, 0.5H2O (585.75): C, 69.72; H, 7.74; N, 4.78; found:
C, 69.54; H, 7.34; N, 4.74. ES-MS (pos. mode): 599.18 [M+Na]+.
4.1.1.5.
sitylene (22).
Tris-[
a
,
a0
,
a00-[para-(Boc-aminoethyl)-phenoxy]]-me-
From A (0.39 g, 1.64 10ꢀ3 mol), K2CO3 (0.23 g,
1.67 10ꢀ3 mol), dry MeCN (30 mL) and mesitylene bromide 21
(0.20 g, 0.56 10ꢀ3 mol) (TLC monitoring; SiO2, CH2Cl2/MeOH
99.8:0.2). Chromatography (SiO2; CH2Cl2/MeOH 99.8:0.2). 22
(0.32 g, 0.39 10ꢀ3 mol, 70%). Mp: 64–66 °C. IR (KBr): 1697.23.
UV–vis (CHCl3): 277 (6428). 1H NMR (400 MHz, CDCl3): 1.43 (s,
27H, Me3C); 2.73 (t, J = 6.80 Hz, 6H, CH2CH2NH); 3.34 (br s, 6H,
CH2CH2NH); 4.55 (br s, 3H, NH); 5.06 (s, 6H, ArCH2O); 6.91 (d,
J = 8.80 Hz, 6H, ArH); 7.10 (d, J = 7.56 Hz, 6H, ArH); 7.46 (s, 3H,
ArH). 13C NMR (100 MHz, CDCl3): 28.43, 35.38, 42.00, 69.78,
79.26, 114.98, 125.94, 129.80, 131.47, 137.99, 155.90, 157.34.
Anal. calcd for C48H63O9N3 (826.03): C, 69.79; H, 7.69; N, 5.09;
found: C, 70.28; H, 7.74; N, 5.15. ES-MS (pos. mode): 848.47
[M+Na] +, 627.31 [Mꢀ2COOCMe3+2H]+.
4.1.1.3.
(12).
Bis-[
From
a,
a0-[para-(Boc-aminoethyl)-phenoxy]-m-xylene
(0.36 g, 1.52 10ꢀ3 mol), K2CO3 (0.21 g,
A
Please cite this article in press as: Massimba-Dibama, H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.053

6
H. Massimba-Dibama et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4.1.1.6.
durene (27).
Tetra-[
a
,
a0
,
a00
,
a000-[para-(Boc-aminoethyl)-phenoxy]]-
From A (0.21 g, 0.89 10ꢀ3 mol), K2CO3 (0.13 g,
5.15 (s, 4H, ArOCH2); 6.89 (d, J = 8.32 Hz, 4H, ArH); 7.12 (d,
J = 8.32 Hz, 4H, ArH); 7.35 (m, 2H, ArH); 7,45 (m, 2H, ArH). 13C
NMR (100 MHz, D2O): 31.84, 40.61, 68.21, 115.44, 129.04,
129.52, 129.98, 130.05, 134.97, 156.84. Anal. calcd for
0.94 10ꢀ3 mol), dry MeCN (20 mL) and 1,2,4,5-tetrabromomethyl
benzene 26 (0.10 g, 0.22 10ꢀ3 mol) (TLC monitoring; SiO2,
CH2Cl2/MeOH 99.8:0.2). Chromatography (SiO2; CH2Cl2/MeOH
97.8:3). 27 (0.23 g, 98%). Mp: 92–94 °C. IR (KBr): 1702.8 (CO).
UV–vis (CH2Cl2): 276.0 (10400). 1H NMR (400 MHz, CDCl3): 1.43
(s, 36H, Me3C); 2.73 (t, J = 6.92 Hz, 8H, CH2CH2NH); 3.33 (br t, 8H,
CH2CH2NH); 4.60 (br s, 4H, NH); 5.15 (s, 8H, ArOCH2); 6.89 (d,
J = 8.56 Hz, 8H, ArH); 7.09 (d, J = 8.56 Hz, 8H, ArH); 7.67 (s, 2H,
ArH). 13C NMR (100 MHz, CDCl3): 28.8, 35.8, 42.4, 68.1, 79.6,
115.3, 130.0, 130.2, 132.0, 135.7, 156.30; 157.6. Anal. calcd for
C24H28O2N2, 2CF3COOH, 2H2O (640.22): C, 52.50; H, 5.35; N,
4.37; found: C, 52.49; H, 5.58; N, 4.13. ES-MS (pos. mode):
377.17 [Mꢀ2CF3COOH+H]+.
4.1.2.3. Bis-[
trifluoroacetate (13).
a
,
a0-[para-(aminoethyl)-phenoxy]-m-xylene, bis
From 12 (0.39 g, 0.68 10ꢀ3 mol) in a
75:25 mixture of CH2Cl2 and TFA (30 mL), 5 h. 13 (0.38 g, 92%).
White solid. Mp: deliquescent. IR (neat):1670.35 (CO). UV–vis
(H2O): 273.0 (2650). 1H NMR (400 MHz, D2O): 2.81 (t, J = 7.30 Hz,
4H, CH2CH2NH); 3.11 (t, J = 7.30 Hz, 4H, CH2CH2NH); 5.03 (s, 4H,
ArOCH2); 6.91 (d, J = 8.56 Hz, 4H, ArH); 7.13 (d, J = 8.56 Hz, 4H,
ArH); 7.34 (s, 3H, ArH); 7.41 (s, 1H, ArH). 13C NMR (100 MHz,
D2O): 31.89, 40.56, 69.47, 115.22, 126.81, 127.33, 128.80, 129.25,
129.88, 136.90, 156.91, 116.39 (q, J = 292 Hz, CF3CO), 162.73 (q,
J = 35 Hz, CF3CO). Anal. calcd for C24H28O2N2, 2CF3COOH (604.20):
C, 55.63; H, 5.00; N, 4.63; found: C, 55.32; H, 5.01; N, 4.70.
ES-MS (pos. mode): 377.20 [Mꢀ2CF3COOH+H]+.
C
62H82O12N4 (1075.33): C, 69.25; H, 7.69; N, 5.21; found: C,
69.00; H, 7.61; N, 5.18. ES-MS (pos. mode): 1097.58 [M+Na]+,
1075.60 [M+H]+.
4.1.1.7. Hexakis-[para-(Boc-aminoethyl)-phenoxymethyl]-ben-
zene (32).
From A (0.22 g, 0.93 10ꢀ3 mol), K2CO3 (0.13 g,
0.94 10ꢀ3 mol), dry MeCN (20 mL) and hexabromomethylbenzene
31 (0.10 g, 0.16 10ꢀ3 mol) (TLC monitoring; SiO2, CH2Cl2/MeOH
99:1). After cooling, the solvent was evaporated to dryness, and
the solid residue was dissolved in CH2Cl2. The solution was washed
with H2O (2 ꢁ 20 mL), the aqueous phase was rinsed with CH2Cl2
(3 ꢁ 15 mL) and the combined organic phases were dried over
Na2SO4, filtered then concentrated prior chromatography (SiO2;
CH2Cl2/MeOH 99.5:0.5). 32 (0.22 g, 97%). Mp: 154–156 °C. IR
(KBr): 1699.77 (CO). UV–vis (CHCl3): 276 (11950). 1H NMR
(400 MHz, CDCl3): 1.43 (s, 54H, Me3C); 2.73 (t, J = 6.80 Hz, 12H,
CH2CH2NH); 3.33 (br s, 12H, CH2CH2NH); 4.60 (br s, 6H, NH);
5.15 (s, 12H, ArCH2O); 6.89 (d, J = 8.30 Hz, 12H, ArH); 7.09 (d,
J = 8.30 Hz, 12H, ArH). 13C NMR (100 MHz, CDCl3): 28.5, 35.5,
42.2, 63.7, 79.4, 114.9, 129.9, 131.9, 138.0, 156.0, 157.2. Anal. calcd
for C90H120O18N6 (1573.95): C, 68.68; H, 7.68; N, 5.34; found: C,
68.37; H, 7.59; N, 5.33. ES-MS (pos. mode): 1596.85 [M+Na]+,
4.1.2.4. Bis-[
fluoroacetate (18).
a
,
a0-[para-(aminoethyl)-phenoxy]-p-xylene, bis tri-
From 17 (1.28 g, 2.22 10ꢀ3 mol), in a
85:15 mixture of CH2Cl2 and TFA (70 mL), 12 h. 18 (1.13 g, 85%).
White solid. Mp: 192–193 °C. IR (KBr): 1676.86 (CO). UV–vis
(H2O): 272 (3203.0). 1H NMR (400 MHz, DMSO-d6): 2.78 (t,
J = 8.1 Hz, 4H, CH2CH2NH), 3.00 (m, 4H, CH2CH2NH), 5.09 (s, 4H,
CH2Ph), 6.97 (d, J = 8.6 Hz, 4H, ArH), 7.17 (d, J = 8.6 H, 4H, ArH),
7.45 (s, 4H, ArH), 7.82 (br s, 2H, NH). 1H NMR (400 MHz, D2O):
2.97 (t, J = 7.1 Hz, 4H, CH2CH2NH), 3.28 (t, J = 8.3 Hz, 4H,
CH2CH2NH), 5.20 (s, 4H, CH2Ph), 7.09 (d, J = 8.0 Hz, 4H, ArH), 7.30
(d, J = 8.6 H, 4H, ArH), 7.54 (s, 4H, ArH). 13C NMR (100 MHz,
D2O): 32.27, 41.06 (CH2CH2NH), 70.47 (OCH2), 116.12, 128.79,
130.04, 130.56, 137.06, 157.33 (C Ar). Anal. calcd for
809.42 [M+2Na]2+/2
.
C28H30F6N2O6, 0.25H2O (609.04): C, 55.22; H, 5.05; N, 4.60; found:
4.1.2. Compounds 3, 8, 13, 18, 23, 28, 33—general procedure
A solution of (Boc-amino) derivative in a mixture of CH2Cl2 and
TFA was stirred at rt under Ar during X h. The solvent was evapo-
rated under vacuum and the residual acid was eliminated by mul-
tiple dissolution/co-evaporation cycles in CH2Cl2 until formation of
a solid material. The latter was treated by multiple trituration–fil-
tration sequences with dry Et2O, until pH of the filtrate remained
neutral, to give the desired trifluoroacetate salt.
C, 55.23; H, 5.07; N, 4.71. ES-MS (pos. mode): 377.20
[Mꢀ2(CF3COOH)+H]+; 360.24 [Mꢀ2(CF3COOH)ꢀNH2+H]+.
4.1.2.5. Tris-[
tris trifluoroacetate (23).
a
,
a0
,
a00-[para-(aminoethyl)-phenoxy]]-mesitylene,
From 22 (0.15 g, 0.18 10ꢀ3 mol) in
a 75:25 mixture of CH2Cl2 and TFA (15 mL), 5 h. After tritura-
tion–filtration sequences with dry Et2O, the white solid was dis-
solved in H2O and the resulting solution was lyophilized to give
23 (0.15 g, 98%). Mp: deliquescent. IR (KBr): 1677.34 (CO). UV–
vis (H2O): 273.0 (4076). 1H NMR (400 MHz, D2O): 2.84 (t,
J = 7.44 Hz, 6H, CH2CH2NH); 3.13 (t, J = 7. 42 Hz, 6H, CH2CH2NH);
4.81 (s, 6H, ArOCH2); 6.84 (t, J = 8.56 Hz, 6H, ArH); 7.13 (t,
J = 7.56 Hz, 6H, ArH); 7.25 (s, 3H, ArH). 13C NMR (100 MHz, D2O):
31.90, 40.57, 69.27, 115.33, 126.45, 129.40, 129.95, 137.49,
156.82, 116.36 (q, J = 292 Hz, CF3CO), 162.80 (CF3CO). Anal. calcd
for C33H39N3O3, 3CF3COOH, H2O (885.29): C, 52.88; H, 5.01; N,
4.74; found: C, 52.96; H, 4.83; N, 4.80. ES-MS (pos. Mode):
526.32 [Mꢀ3CF3COOH+H]+.
4.1.2.1.
etate (3).
a
-[para-(Aminoethyl)-phenoxy]toluene, trifluoroac-
From 2 (0.10 g, 0.30 10ꢀ3 mol) in a 75:25 mixture
of CH2Cl2 and TFA (15 mL), 5 h. 3 (0.10 g, 96%). White powder.
Mp: 148–150 °C. IR (KBr): 3037.75 (NH+3); 1676.29, 1634.02 (CO).
UV–vis (D2O): 273 (1641.9). 1H NMR (400 MHz, D2O): 2.89 (t,
J = 7.04 Hz, 2H, ArCH2CH2NH); 3.19 (t, J = 7.18 Hz, 2H,
ArCH2CH2NH); 5.13 (s, 2H, ArOCH2); 7.02 (d, J = 8.56 Hz, 2H,
ArH); 7.22 (d, J = 8.28 Hz, 2H, ArH); 7.37–7.47 (m, 5H, ArH). 13C
NMR (100 MHz, DMSO-d6): 32.57, 48.13, 69.51, 115.30, 127.97,
128.16, 128.80, 130.10, 137.51, 147.51, 157.16. Anal. calcd for
C15H17NO, CF3COOH, 0.25H2O (345.82): C, 59.04; H, 5.39; N, 4.05;
4.1.2.6.
Tetra-[
a
,
a0
,
a00
,
a000-[para-(aminoethyl)-phenoxy]]-dur-
From 27 (0.22 g,
found: C, 59.37, H, 5.29, N, 4.09. ES-MS (pos. mode): 228.05
ene, tetra trifluoroacetate (28).
[MꢀCF3COOH+H]+, 210.72 [MꢀCF3COOHꢀNH2]+.
0.20 10ꢀ3 mol) in a 75:25 mixture of CH2Cl2 and TFA (20 mL),
24 h. 28 (0.19 g, 80%). White solid. Mp: deliquescent. IR (KBr):
1681.42 (CO). UV–vis (H2O): 273.0 (6050). 1H NMR (400 MHz,
DMSO-d6): 2.79 (br t, 8H, CH2CH2NH); 2.99 (br t, 8H,
CH2CH2NH); 5.20 (s, 8H, ArOCH2); 6.98 (d, J = 8.81 Hz, 8H, ArH);
7.17 (d, J = 8.80 Hz, 8H, ArH); 7.68 (s, 2H, ArH), 7.98 (br s, 12H,
NH+3). 13C NMR (100 MHz, DMSO-d6): 32.3, 40.2, 66.8, 115.0,
128.9, 129.7, 129.8, 135.1, 157.1, 157.1, 158.4 (q, J = 30.8 Hz).
4.1.2.2. Bis-[
trifluoroacetate (8).
a
,
a0-[para-(aminoethyl)-phenoxy]-o-xylene, bis-
From
7
(0.26 g, 0.44 10ꢀ3 mol) in
a
75:25 mixture of CH2Cl2 and TFA (20 mL), 5 h. 8 (0.20 g, 71%).
White solid. Mp: 127–128 °C. IR (KBr): 1678.40 (CO). UV–vis
(H2O): 273.0 (2970.1). 1H NMR (400 MHz, D2O): 2.81 (t,
J = 7.18 Hz, 4H, CH2CH2NH); 3.11 (t, J = 7.18 Hz, 4H, CH2CH2NH);
Please cite this article in press as: Massimba-Dibama, H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.053

H. Massimba-Dibama et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
Anal. calcd for C42H50O4N4,4 CF3COOH, H2O (1151.0): C, 52.18; H,
5.08; N, 4.87; found: C, 51.94; H, 4.84; N, 4.87. ES-MS (pos. mode):
789.3 [Mꢀ3CF3COOH+H]+; 675.39 [Mꢀ4CF3COOH+H]+.
found: C, 60.49; H, 6.95; N, 9.61. ES-MS (pos. mode): 883.43
[M+Na]+, 861.53 [M+H]+, 761.42 [MꢀCOOCMe3+2H]+, 431.00
[M+2H]2+/2
.
4.1.2.7. Hexakis-[para-(aminoethyl)-phenoxymethyl]benzene
(33).
4.1.3.3.
xylene (19).
Bis-[
a,
a0-[para-(di-Boc-guanidinoethyl)-phenoxy]-p-
From 18 (1.00 g, 1.65 10ꢀ3 mol), dry CH2Cl2
From 32 (0,20 g, 0.13 10ꢀ3 mol) in a 75:25 mixture of
CH2Cl2 and TFA (20 mL), 3 h. 33 (0.19 g, 88%). White solid. Mp: del-
iquescent. IR (KBr): 1677.1 (ꢀNH+3). UV–vis (H2O): 272 (8985). 1H
NMR (400 MHz, D2O): 2.87 (t, J = 7.30 Hz, 12H, ArCH2CH2NH);
3.17 (t, J = 7.42 Hz, 12H, ArCH2CH2NH); 5.35 (s, 12H, ArCH2O);
6.91 (d, J = 8.56 Hz, 12H, ArH); 7.14 (d, J = 8.80 Hz, 12H, ArH). 13C
NMR (100 MHz, D2O): 31.9, 40.6, 64.3, 115.2, 129.6, 129.7, 137.8,
156.9. Anal. calcd for C60H72N6O6, 6 CF3COOH, 2H2O (1692.5): C,
51.07; H, 4.67; N, 4.96; found: C, 51.12; H, 4.67; N, 4.96. ES-MS
(50 mL), MeOH (5 mL), Et3N (1.38 mL, 9.92 10ꢀ3 mol), B (1.36 g,
3.47 10ꢀ3 mol), 7 h (TLC monitoring SiO2, CH2Cl2/MeOH 99.9:0.1).
Residue of evaporation dissolved in CH2Cl2 (50 mL), washed with
H2O (30 mL). Chromatography (SiO2, CH2Cl2). 19 (1.23 g, 87%).
White solid. Mp: 104–105 °C. IR (pure): 1714.72, 1610.56 (CO).
UV–vis (CH2Cl2): 275 (2244). 1H NMR (400 MHz, CDCl3): 1.48 (s,
18H, Me3C), 1.50 (s, 18H, Me3C), 2.82 (t, J = 7.3 Hz, 4H,
CH2CH2NH), 3.64 (q, J = 6.8 Hz, 4H, CH2CH2NH), 5.05 (s, 4H,
CH2Ph), 6.90 (d, J = 8.6 Hz, 4H, ArH), 7.13 (d, J = 8.6 H, 4H, ArH),
7.44 (s, 4H, ArH), 8.37 (s large, 2H, NH), 11.46 (s, 2H, NH). 13C
NMR (100 MHz, CDCl3): 28.43, 28.71, 34.77, 42.85, 70.10, 79.64,
83.40, 115.33, 128.05, 130.19, 131.28, 137.22, 153.53, 156.54,
157.85, 163.95. Anal. calcd for C46H64N6O10 (861.03): C, 64.17; H,
7.49; N, 9.76; found: C, 64.17; H, 7.54; N, 9.79. ES-MS (pos. mode):
899.44 [M+K]+, 883.46 [M+Na]+, 861.48 [M+H]+, 761.42
(pos.
mode):
487.27
[Mꢀ6CF3COOH+2H]2+/2
;
325.19
[Mꢀ6CF3COOH+3H]3+/3; 308.17 [Mꢀ6CF3COOHꢀ3NH2+3H]3+/3
.
4.1.3. Compounds 4, 14, 19, 24, 29, 34—general procedure
The ammonium salts were suspended in dry CH2Cl2, and solubi-
lized with the minimum of MeOH; Et3N was added, then
N,N0-bis(tert-butoxycarbonyl)-N00-triflylguanidine B. The mixture
was stirred at rt under Ar during X h (TLC monitoring, SiO2,
CH2Cl2/MeOH, X:Y%). After evaporation of the solvent, the residue
was dissolved in CH2Cl2, and the solution was washed with H2O;
the aqueous phase was washed with CH2Cl2, and the combined
organic phases were dried over Na2SO4, filtered and evaporated
to dryness. The resulting solid was chromatographed (SiO2,
CH2Cl2/MeOH, gradient) to give the desired –(di-Boc-guanidinoet
hyl)– derivatives.
[MꢀCOOCMe3+2H]+, 431.21 [M+2H]2+/2
.
4.1.3.4. Tris-[
mesitylene (24).
a
,
a0
,
a00-[para-(di-Boc-guanidinoethyl)-phenoxy]]-
From 23 (0.14 g, 0.16 10ꢀ3 mol), dry CH2Cl2
(5 mL), MeOH (3 mL), Et3N (0.27 mL, 1.93 10ꢀ3 mol), B (0.19 g,
0.49 10ꢀ3 mol), 24 h (TLC monitoring, SiO2, CH2Cl2/MeOH,
99.9:0.1). Residue of evaporation dissolved in CH2Cl2 (25 mL),
washed with H2O (10 mL); washing of aqueous phase with
CH2Cl2 (3 ꢁ 10 mL). Chromatography (SiO2, CH2Cl2). 24 (0.18 g,
89.9%). White solid. Mp: 80–82 °C. IR (KBr): 1721.31, 1638.71
(CO). UV–vis (CH2Cl2): 276 (7220). 1H NMR (400 MHz, CDCl3):
1.47 (s, 27H, Me3C); 1.50 (s, 27H, Me3C); 2.81 (t, J = 7.30 Hz, 6H,
CH2CH2NH); 3.63 (t, J = 6.53 Hz, 6H, CH2CH2NH); 5.06 (s, 6H,
ArOCH2); 6.91 (d, J = 8.56 Hz, 6H, ArH); 7.13 (d, J = 7.80 Hz, 6H,
ArH); 7.47 (s, 3H, ArH); 8.36 (t, J = 4.78 Hz, 3H, CH2NH); 11.46 (s,
3H, NH). 13C NMR (100 MHz, CDCl3): 28.46, 28.72, 34.83, 42.85,
70.18, 79.64, 83.41, 115.32, 126.40, 130.22, 131.42, 138.34,
138.40, 153.57, 156.52, 157.83, 164.02. Anal. calcd for
4.1.3.1.
(4).
a
-[para-(di-Boc-guanidinoethyl)-phenoxy]toluene
From 3 (0.10 g, 0.29 10ꢀ3 mol), dry CH2Cl2 (10 mL), Et3N
(112 l
L, 0.87 10ꢀ3 mol), B (0.114 g, 0.29 10ꢀ3 mol), 9 h (TLC moni-
toring, SiO2, CH2Cl2/MeOH, 99.9:0.1). Residue of evaporation dis-
solved in CH2Cl2 (30 mL), washed with H2O (30 mL); washing of
aqueous phase with CH2Cl2 (2 ꢁ 20 mL). Chromatography (SiO2,
CH2Cl2/MeOH, 100:0 to 99.5:0.5). 4 (0.12 g, 85%). White solid.
Mp: 99–100 °C. IR (KBr): 1730.4 (CO). UV–vis (CDCl3): 276
(1577.5). 1H NMR (400 MHz, CDCl3): 1.48 (s, 9H, Me3C); 1.51 (s,
9H, Me3C); 2.81 (t, J = 7.18 Hz, 2H, ArCH2CH2NH); 3.64 (m, 2H,
CH2CH2NH); 5.04 (s, 2H, ArOCH2); 6.91 (d, J = 8.56 Hz, 2H, ArH);
7.13 (d, J = 8.56 Hz, 2H, ArH); 7.37–7.43 (m, 5H, ArH); 8.37 (br t,
1 H, NH); 11.49 (br s, 1 H, NHguan). 13C NMR (100 MHz, CDCl3):
28.46, 28.74, 34.80, 42.85, 70.42, 79.61, 83.38, 115.36, 127.87,
128.32, 128.97, 130.19, 131.29, 137.52, 153.56, 156.55, 157.93,
164.03. Anal. calcd for C26H35O5N3, 0.1 C(NH)(NH2)2, 0.3 CH2Cl2
(500.96): C, 63.20; H, 7.23; N, 9.26; found: C, 63.29; H, 7.26; N,
9.23. ES-MS (pos. mode): 319.11 [MꢀCOOCMe3ꢀMe3CO+Na+H]+.
C66H93O15N9, 0.5CH2Cl2 (1335.63): C, 61.68; H, 7.32; N, 9.73;
found: C, 61.67; H, 7.24; N, 9.89. ES-MS (pos. mode): 1252.67
[M+H]+; 1274.66 [M+Na]+; 1290.61 [M+K]+.
4.1.3.5.
noxy]]-durene (29).
Tetra-[
a
,
a0
,
a00
,
a000-[para-(di-Boc-guanidinoethyl)-phe-
From 28 (0.18 g, 0.16 10ꢀ3 mol), dry
CH2Cl2 (10 mL), MeOH (10 mL), Et3N (0.28 mL, 2.00 10ꢀ3 mol), B
(0.26 g, 0.66 10ꢀ3 mol), 24 h (TLC monitoring, SiO2, CH2Cl2/MeOH,
99.9:0.1). Residue of evaporation dissolved in CH2Cl2 (20 mL),
washed with H2O (2 ꢁ 20 mL); washing of aqueous phase with
CH2Cl2 (3 ꢁ 20 mL). Chromatography (SiO2, CH2Cl2/MeOH, 100:0
to 99:1). 29 (0.16 g, 61%). White powder. Mp: 134–136 °C. IR
(KBr): 1638.74, 1721.54 (CO). UV–vis (CH2Cl2): 276 (9980). 1H
NMR (400 MHz, CDCl3): 1.46 (s, 36H, Me3C); 1.49 (s, 36H, Me3C);
2.80 (t, J = 7.16 Hz, 8H, CH2CH2NH); 3.61 (m, 8H, CH2CH2NH);
5.13 (s, 8H, ArOCH2); 6.88 (d, J = 8.08 Hz, 8H, ArH); 7.11 (d,
J = 7.84 Hz, 8H, ArH); 7.67 (s, 2H, ArH); 8.37 (br s, 4H, NH); 11.30
(br s, 4H, NHguan). 13C NMR (100 MHz, CDCl3): 28.4, 28.6, 34.7,
42.7, 67.9, 79.6, 83.3, 115.2, 129.9, 130.2, 131.5, 135.5, 153.5,
156.4, 157.6, 163.9. Anal. calcd for C86H122O20N12 (1643.96): C,
62.83; H, 7.48; N, 10.22; found: C, 62.79; H, 7.50; N, 10.02.
ES-MS (pos. mode): 1643.88 [M+H]+; 1681.83 [M+K]+; 1666.85
4.1.3.2.
xylene (14).
Bis-[
a
,
a0-[para-(di-Boc-guanidinoethyl)-phenoxy]-m-
From 13 (0.17 g, 0.28 10ꢀ3 mol), dry CH2Cl2
(15 mL), MeOH (0.3 mL), Et3N (250 l
L, 1.66 10ꢀ3 mol), B (0.22 g,
0.56 10ꢀ3 mol), 24 h (TLC monitoring, SiO2, CH2Cl2/MeOH,
99.9:0.1). Residue of evaporation dissolved in CH2Cl2 (50 mL),
washed with H2O (40 mL); washing of aqueous phase with
CH2Cl2 (2 ꢁ 20 mL). Chromatography (SiO2, CH2Cl2). 14 (0.23 g,
98.8%). White solid. Mp: 98–100 °C. IR (KBr): 1721.70, 1638.94
(CO). UV–vis (CH2Cl2) 276 (2746.31). 1H NMR (400 MHz, CDCl3):
1.48 (s, 18H, Me3C); 1.50 (s, 18H, Me3C); 2.81 (t, J = 7.30 Hz, 4H,
ArCH2CH2NH2); 3.62 (m, 4H, ArCH2CH2NH2); 5.05 (s, 4H,
ArOCH2); 6.91 (d, J = 8.52 Hz, 4H, ArH); 7.13 (d, J = 8.56 Hz, 4H,
ArH); 7.39 (br s, 3H, ArH); 7.50 (s, 1 H, ArH); 8.36 (br s, 2H, NH);
11.47 (s, 2H, NHguan). 13C NMR (100 MHz, CDCl3): 28.44, 28.70,
34.77, 42.87, 70.29, 79.64, 83.41, 115.33, 126.87, 127.43, 129.24,
130.20, 131.32, 137.89, 153.54, 156.57, 157.87, 163.92. Anal. calcd
for C46H64O10N6, 0.75 CH2Cl2 (924.17): C, 60.75; H, 7.14; N, 9.10;
[M+Na]+; 822.9 [M+2H]2+/2
.
4.1.3.6.
noxymethyl]benzene
0.106 10ꢀ3 mol), dry CH2Cl2 (10 mL), MeOH (10 mL), Et3N
Hexakis-[para-(di-Boc-guanidinoethyl)-phe-
(34). From 33 (0.18 g,
Please cite this article in press as: Massimba-Dibama, H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.053

8
H. Massimba-Dibama et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
(0.28 mL, 2.00 10ꢀ3 mol), B (0.26 g, 0.66 10ꢀ3 mol), 15 h (TLC mon-
itoring, SiO2, CH2Cl2/MeOH, 99.9:0.1). Residue of evaporation dis-
solved in CH2Cl2 (20 mL), washed with H2O (2 ꢁ 25 mL); washing
of aqueous phase with CH2Cl2 (3 ꢁ 10 mL). Chromatography
(SiO2, CH2Cl2/MeOH, 99:1). 34 (0.22 g, 84%). White powder. Mp:
152–154 °C IR (KBr): 1721.91, 1638.39, 1615.16 (CO). UV
(CH2Cl2): 275.0 (10486). 1H NMR (400 MHz, CDCl3): 1.45 (s, 54H,
Me3C); 1.49 (s, 54H, Me3C); 2.79 (t, J = 6.80 Hz, 12H,
ArCH2CH2NH); 3.60 (br t, 12H, ArCH2CH2NH); 5.15 (s, 12H,
ArCH2O); 6.81 (d, J = 8.08 Hz, 12H, ArH), 7.06 (d, J = 8.04 Hz, 12H,
ArH); 8.39 (br s, 6H, NH); 11.46 (br s, 6H, NHguan). 13C NMR
(100 MHz, CDCl3): 28.04; 28.31; 34.46; 42.49; 63.64; 79.30;
83.05; 114.81; 129.84; 131.31; 137.88; 153.15; 156.06;157.21;
163.48. Anal. calcd for C126H180O30N18, 0.3CH2Cl2 (2452.36): C,
61.86; H, 7.42; N, 10.28; found: C, 61.83; H, 7.07; N, 10.50.
ES-MS (pos. mode): 2426.32 [M+H]+.
4.1.4.2. Bis-[
trifluoroacetate (10).
a
,
a0-[para-(guanidinoethyl)-phenoxy]-o-xylène, bis
From 9 (0.50 g, 0.58 10ꢀ3 mol) in a
75:25 mixture of CH2Cl2 and TFA (30 mL), 24 h. 10 (0.30 g,
75.6%). White solid. Mp: deliquescent. IR (pure): 1671.9 (CO).
UV–vis (H2O): 272 (3394). 1H NMR (400 MHz, D2O): 2.80 (t,
J = 6.42 Hz, 4H, CH2CH2NH); 3.39 (t, J = 6.66 Hz, 4H, CH2CH2NH);
5.24 (s, 4H, ArOCH2); 6.96 (d, J = 8.32 Hz, 4H, ArH); 7.18 (d,
J = 8.56 Hz, 4H, ArH); 7.45 (m, 2H, ArH); 7.54 (m, 2H, ArH). 13C
NMR (100 MHz, D2O): 33.55, 42.74, 68.12, 115.25, 128.86,
129.58, 130.18, 131.32, 135.21, 156.96, 157.01. Anal. calcd for
C26H32O2N6, 2 CF3COOH, H2O (706.63): C, 50.99; H, 5.14; N,
11.89; found: C, 51.09; H, 4.92; N, 11.54. ES-MS (pos. mode):
461.26 [Mꢀ2CF3COOH+H]+, 230.82 [Mꢀ2CF3COOH+2H]2+/2
.
4.1.4.3. Bis-[
bis trifluoroacetate (15).
a,
a0-[para-(guanidinoethyl)-phenoxy]]-m-xylene,
From 14 (0.15 g, 0.17 10ꢀ3 mol) in
a 75:25 mixture of CH2Cl2 and TFA (20 mL), 5 h. 15 (0.12 g,
0.17 10ꢀ3 mol, 98%). White solid. Mp: deliquescent. IR (neat):
1672.28, 1654.92, 1624.06, 1616.35 (CO). UV–vis (H2O): 273.0
(3302.0). 1H NMR (400 MHz, D2O): 2.69 (t, J = 6.56 Hz, 4H,
CH2CH2NH); 3.27 (t, J = 6.66 Hz, 4H, CH2CH2NH); 4.94 (s, 4H,
ArOCH2); 6.82 (d, J = 8.28 Hz, 4H, ArH); 7.06 (d, J = 8.32 Hz, 4H,
ArH); 7.29 (br s, 3H, ArH); 7.32 (br s, 1H, ArH). 13C NMR
(100 MHz, D2O): 33.55; 42.74; 68.12; 115.25; 128.86; 129.58;
130.18; 131.32; 135.21; 156.96; 157.01; 165.21. Anal. calcd for
4.1.3.7.
xylene (9).
Bis-[
a
,
a0-[para-(di-Boc-guanidinoethyl)-phenoxy]-o-
The salt 8 (0.30 g, 4.70 10-4 mol) was solubilised
in anhydrous DMF (30 mL), then Et3N (1.11 mL, 7.95 10ꢀ3 mol)
and the 1,3-bis-(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea
C (0.73 g, 2.50 10ꢀ3 mol) were added. The mixture was stirred
under Ar at 4 °C, then HgCl2 (0.68 g, 2.50 10ꢀ3 mol. Warning:
HgCl2 must be handled with care) was added in one portion.
After stirring at rt under Ar overnight, the solvent was evaporated
under high vacuum at 30 °C. The resulting solid was dissolved in
CH2Cl2 and the insoluble material was filtered off over Celite. The
filtrate was concentrated then chromatographed (Al2O3,
CH2Cl2/MeOH 99.8/0.2) to give 9 (0.39 g, 94%). White powder.
Mp: 85–86 °C. IR (KBr): 1721.70, 1638.94 (CO). UV–vis (CH2Cl2):
276.0 (5382.4). 1H NMR (400 MHz, CDCl3): 1.47 (s, 18H, Me3C);
1.50 (s, 18H, Me3C); 2.81 (t, J = 7.30 Hz, 4H, CH2CH2NH2); 3.64 (q,
J = 6.90 Hz, 4H, CH2CH2NH); 5.14 (s, 4H, ArOCH2); 6.90 (d,
J = 8.52 Hz, 4H, ArH); 7.12 (d, J = 8.56 Hz, 4H, ArH); 7.35 (m, 2H,
ArH); 7.51 (m, 2H, ArH); 8.39 (br s, 2H, NH); 11.33 (br s, 2H,
NHguan). 13C NMR (100 MHz, CDCl3): 28.04, 28.31, 34.40, 42.55,
67.99, 79.44, 83.13, 114.96, 128.37, 128.86, 129.83, 131.00,
135.17, 153.14, 156.06, 157.38, 163.99. Anal. calcd for
C26H32O2N6, 2CF3COOH, 1/2H2O (697.63): C, 51.65; H, 5.06; N,
12.05; found: C, 51.60; H, 4.86, N12.06. ES-MS (pos. mode):
461.25 [Mꢀ2CF3COOH+H]+; 231.05 [Mꢀ2CF3COOH+2H]2+/2
.
4.1.4.4.
bis trifluoroacetate (20).
Bis-[
a
,
a0-[para-(guanidinoethyl)-phenoxy]]-p-xylene,
From 19 (0.40 g , 0.46 10ꢀ3 mol),
in a 80:20 mixture of CH2Cl2 and TFA (25 mL), 24 h. 20 (0.25 g,
79%). White powder. Mp: deliquescent. IR (pure): 1612.49,
1629.85 (CO). UV–vis (H2O): 273 (3032). 1H NMR (400 MHz,
D2O): 2.89 (t, J = 6.8 Hz, 4H, CH2CH2NH), 3.47 (t, J = 6.8 Hz, 4H,
CH2CH2NH), 5.22 (s, 4H, CH2Ph), 7.06 (d, J = 8.8 Hz, 4H, ArH), 7.29
(d, J = 8.8 H, 4H, ArH), 7.57 (s, 4H, ArH). 1H NMR (400 MHz,
DMSO-d6): 2.70 (t, J = 7.1 Hz, 4H, CH2CH2NH), 3.30 (q, J = 6.5 Hz,
4H, CH2CH2NH), 5.07 (s, 4H, CH2Ph), 6.94 (d, J = 8.3 Hz, 4H, ArH),
7.17 (d, J = 8.3H, 4H, ArH), 7.22 (br s, 8H, NH), 7.44 (s, 4H, ArH),
7.66 (t, J = 4.9 Hz, 2H, CH2CH2NH). 13C NMR (100 MHz,
DMSO-d6): 36.35, 45.04 (CH2CH2NH), 71.70 (OCH2), 117.51,
130.50, 132.58, 133.24, 139.56, 159.61, 159.75 (C Ar and
Cguanidine). Anal. calcd for C26H32O2N6, 2CF3COOH (688.62): C,
52.33; H, 4.98; N, 12.20; found: C, 52.37; H, 5.05; N, 12.16.
ES-MS (pos. mode): 461.27 [Mꢀ2CF3COOH+H]+.
C
46H64O10N6, 1.5H2O (887.49): C, 62.25; H, 7.61; N, 9.47; found:
C, 61.96; H, 7.35; N, 9.80. ES-MS (pos. mode): 861.6 [M+H]+,
761.4 [MꢀCOOCMe3+H]+, 431.4 [M+2H]2+/2
.
4.1.4. Compounds 5, 10, 15, 20, 25, 30, 35—general procedure
A solution of (di-Boc-guanidino) derivative in a mixture of
CH2Cl2 and TFA was stirred at rt under Ar during X h. The solvent
was evaporated under vacuum and the residual acid was elimi-
nated by multiple dissolution/co-evaporation cycles in CH2Cl2 until
formation of a solid material. The latter was treated by multiple tri
turation–ultrasonication–filtration sequences with dry Et2O, until
pH of the filtrate remained neutral. The solid material was dis-
solved in H2O and lyophilized to give the desired trifluoroacetate
salt.
4.1.4.5.
Tris-[
a
,
a0
,
a00-[para-(guanidinoethyl)-phenoxy]]-me-
From 24 (0.16 g,
sitylene, tris trifluoroacetate (25).
0.12 10ꢀ3 mol) in a 75:25 mixture of CH2Cl2 and TFA (15 mL),
6 h. (TLC monitoring; SiO2, CH2Cl2/CH3OH 2%). 25 (0.11 g, 90%).
Mp: deliquescent. IR (KBr): 1670.4 (CO). UV–vis (H2O): 274
(4050). 1H NMR (400 MHz, D2O): 2.76 (t, J = 6.04 Hz, 6H,
CH2CH2NH); 3.34 (t, J = 6.04 Hz, 6H, CH2CH2NH); 4.89 (s, 6H,
ArOCH2); 6.85 (t, J = 7.32 Hz, 6H, ArH); 7.12 (t, J = 7.56 Hz, 6H,
ArH); 7.29 (s, 3H, ArH). 13C NMR (100 MHz, D20): 33.16, 42.40,
69.02, 114.95, 126.05, 129.78, 130.87, 137.39, 156.57, 156.64,
116.4 (q, J = 292 Hz, CF3CO), 162.46 (CF3CO). Anal. calcd for
4.1.4.1.
etate (5).
a
-[para-(Guanidinoethyl)-phenoxy]toluene, trifluoroac-
From 4 (0.150 g, 0.32 10ꢀ3 mol) in a 75:25 mixture
of CH2Cl2 and TFA (15 mL), 6 h. 5 (0.12 g, 94%). White powder. Mp:
160–162 °C. IR (KBr): 1664.5 (CO). UV–vis (H2O): 274.0 (1026.00).
1H NMR (400 MHz, D2O): 2.81 (t, J = 6.68 Hz, 2H, CH2CH2NH); 3.40
(t, J = 6.68 Hz, 2H, CH2CH2NH); 5.15 (s, 2H, ArOCH2); 7.02 (d,
J = 8.56 Hz, 2H, ArH); 7.22 (d, J = 8.28 Hz, 2H, ArH); 7.38–7.46 (m,
5H, ArH). 13C NMR (100 MHz, DMSO-d6): 33.91, 42.62, 69.50,
115.05, 127.98, 128.13, 128.78, 130.16, 130.78, 137.55, 157.15,
157.34. Anal. calcd for C16H19ON3, CF3COOH, 0.5H2O (392,37): C,
55.10; H, 5.39; N, 10.71; found: C, 55.03; H, 5.43; N, 10.97.
ES-MS (pos. mode): 270.28 [MꢀCF3COOH+H]+.
C36H45N9O3, 3CF3COOH, 0.5H2O (1002.88): C, 50.30; H, 4.92; N,
12.57; found: C, 50.16; H, 4.97; N, 12.46. ES-MS (pos. mode): 880
[MꢀCF3COOH+H]+; 766 [Mꢀ2CF3COOH+H]+.
4.1.4.6.
Tetra-[a, , ,
a0 a00 a000-[para-(guanidinoethyl)-phenoxy]]-
durene, tetra trifluoroacetate (30).
From 29 (0.14 g,
0.08 10ꢀ3 mol) in a 75:25 mixture of CH2Cl2 and TFA (15 mL),
24 h. (TLC monitoring; SiO2, CH2Cl2/CH3OH 99:1). 30 (0.08 g,
Please cite this article in press as: Massimba-Dibama, H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.053

H. Massimba-Dibama et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
71%). White solid. Mp: deliquescent. IR (KBr): 1671.14 (CO). UV–vis
(H2O): 272 (6126). 1H NMR (400 MHz, D2O): 2.74 (t, J = 6.78 Hz, 8H,
CH2CH2NH); 3.31 (t, J = 6.92 Hz, 8H, CH2CH2NH); 5.01 (s, 8H,
ArOCH2); 6.82 (d, J = 8.32 Hz, 8H, ArH); 7.08 (d, J = 8.28 Hz, 8H,
ArH); 7.49 (s, 2H, ArH). 13C NMR (100 MHz, DMSO-d6): 33.6, 42.3,
66.8, 114.7, 128.8, 129.9, 130.7, 135.1, 156.8, 156.9, 158.9 (q,
J = 31.3 Hz). Anal. calcd for C46H58O4N12, 4 CF3COOH, 1.5H2O
(1326.15): C, 48.91; H, 4.94; N, 12.67; found: C, 49.00; H, 5.27;
N, 12.63. ES-MS (pos. mode): 1185.47 [MꢀCF3COOH+H]+, 1071.48
5.0 software. The reported MICs are an average of at least three
individual measurements.
Acknowledgments
We are grateful to the MRES, the CNRS and the Region Lorraine
for financial support. The authors thank F. Dupire and S. Adach for
mass measurements and elemental analyses, and B. Fernette for
NMR analyses. We express our sincere gratitude to Prof. Brigitte
Gicquel and Véronique Cadet-Daniel (Institut Pasteur, Paris,
France) and Catherine Pierre-Audigier (Hospital Bichat, Paris,
France) for the gift of the INH-R strain.
[Mꢀ2CF3COOH+H]+,
[Mꢀ4CF3COOH+H]+.
957.48
[Mꢀ3CF3COOH+H]+,
843.49
4.1.4.7. Hexakis-[para-(guanidinoethyl)-phenoxymethyl]ben-
zene, hexa trifluoroacetate (35). From 34 (0.14 g,
References and notes
0.06 10ꢀ3 mol) in a 75:25 mixture of CH2Cl2 and TFA (20 mL),
24 h. (TLC monitoring; SiO2, CH2Cl2/CH3OH 98:2). 35 (0.09 g,
79%). White solid. Mp: deliquescent. IR (KBr): 1670.26 (CO). UV–
vis (H2O): 273 (1350.0). 1H NMR (400 MHz, D2O): 2.77 (t,
J = 6.80 Hz, 12H, ArCH2CH2NH); 3.35 (t, J = 6.80 Hz, 12H,
ArCH2CH2NH); 5.33 (s, 12H, ArOCH2); 6.87 (d, J = 8.56 Hz, 12H,
ArH); 7.11 (d, J = 8.56 Hz, 12H, ArH). 13C NMR (100 MHz, D2O):
33.37; 42.54; 64.56; 115.25; 130.05; 131.66; 138.05; 156.72;
158.80. (no visible CF3COOH). Anal. calcd for C66H84N18O6, 6
CF3COOH, 2.5H2O (1954.66): C, 47.93; H, 4.90; N, 12.90; found C
47.91; H, 5.05; N, 12.86. ES-MS (pos. mode): 613.3
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;
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Please cite this article in press as: Massimba-Dibama, H.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.053