Parallel synthesis of chiral pentaamines and pyrrolidine containing bis-heterocyclic libraries. Multiple scaffolds with multiple building blocks: A double diversity for the identification of new antitubercular compounds

Article abstract of DOI:10.1016/j.bmcl.2009.07.010

Combinatorial chemistry offers a unique opportunity for the synthesis and screening of large numbers of compounds and significantly enhances the prospect of finding new drugs. Collaborative efforts with the Tuberculosis Antimicrobial Acquisition & Coordin

Full text of DOI:10.1016/j.bmcl.2009.07.010

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5169–5175
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Parallel synthesis of chiral pentaamines and pyrrolidine containing
bis-heterocyclic libraries. Multiple scaffolds with multiple building blocks: A
double diversity for the identification of new antitubercular compounds
*
Adel Nefzi , Jon Appel, Sergey Arutyunyan, Richard A. Houghten
Torrey Pines Institute for Molecular Studies, 3550 Genearl Atomocs Court, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Combinatorial chemistry offers a unique opportunity for the synthesis and screening of large numbers of
compounds and significantly enhances the prospect of finding new drugs. Collaborative efforts with the
Tuberculosis Antimicrobial Acquisition & Coordinating Facility (TAACF), have led to the identification of
submicromolar novel antitubercular hits. Chiral pentaamines and bis-heterocyclic compounds with 90–
100% inhibition against Mycobacterium tuberculosis strain H37Rv were identified. Some of the identified
compounds are more active than the existing drug ethambutol.
Received 8 May 2009
Revised 22 June 2009
Accepted 2 July 2009
Available online 9 July 2009
Keywords:
Pentaamines
Ó 2009 Published by Elsevier Ltd.
Pyrrolidine bis-heterocyclic compounds
Cyclic guanidines
Cyclic thioureas
Piperazines
Diketopiperazines
Antitubercular activity
Tuberculosis (TB) is the biggest reemerging infectious disease in
recent history and kills an estimated 3 million people worldwide
each year. Although many believe TB to be a scourge of the past,
the disease continues to strike people throughout the world at an
alarming rate. Almost 2 billion people are infected with Mycobacte-
rium tuberculosis, the TB bacterium, and each year, 8 million of
them develop active TB.1–3 Tuberculosis is particularly dangerous
to those with weakened immune systems and is the primary cause
of death among people infected with HIV.4 In recent years, drug-
resistant strains of TB have emerged, posing a formidable threat
to the global population. The discovery and development of new
therapeutic anti-TB regimens compounds are urgently needed.
Combining the power of combinatorial chemistry and close collab-
orative interactions with the Tuberculosis Antimicrobial Acquisi-
tion & Coordinating Facility (TAACF),5 a leading laboratory having
assays and screening facilities against tuberculosis, a large selec-
tion of diversified compounds were tested and led to the identifi-
cation of potential useful lead compounds. Low molecular weight
compounds, especially heterocyclic compounds, offer a high de-
gree of structural diversity and have proven to be broadly and eco-
nomically useful as therapeutic agents.6–11 We have proven the
inherent strengths of combinatorial chemistry (large numbers of
individual compounds and mixture based combinatorial libraries)
to accelerate chemical information acquisition for basic research
and drug design.12,13
Polyamines were reported to have high antitubercular activi-
ties.14 It has been recently reported that active chiral diamines
were identified following the screening of a large library of com-
pounds.14 It has been described that amines which occurred most
frequently in active compounds included many with large hydro-
phobic moieties, suggesting that optimization was perhaps select-
ing for the isoprenoid binding site of the arabinosyltransferase
target of the antitubercular drug ethambutol (EMB) (Fig. 1).
Encouraged by the reported antitubercular activities of poly-
amines, we sent 120 individual chiral pentaamines to TACFF. As
HO
H
N
N
H
Abbreviations: SPPS, solid phase peptide synthesis; PS-SCL, positional scanning
synthetic combinatorial library; TB, tuberculosis; MIC, minimal inhibitory concen-
tration; TAACF, Tuberculosis Antimicrobial Acquisition & Coordinating Facility.
* Corresponding author. Tel.: +1 858 597 3803; fax: +1 858 597 3804.
E-mail address: adeln@tpims.org (A. Nefzi).
OH
Ethambutol (EMB)
MIC = 10 µg/ml
Figure 1. Chemical structure of ethambutol.
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.07.010

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A. Nefzi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5169–5175
R2
O
O
R1
O
H
N
H
N
N
H
R4
N
N
H
R3
O
O
BH3-THF
R2
R2
R1
H
N
1) C2O2Im2
R4
2) BH3-THF
R2
N
H
N
N
R1
H
N
N
HF/anisole
H
N
N
R4
N
H
H2N
R3
HN
N
H
N
N
H
R3
S
3) HF/anisole
1) CSIm2
2) HF/anisole
R1
N
1) C2O2Im2
2) HF/anisole
R3
R4
1) CNBr
TPI-1345
O
TPI-1343
R2
O
O
R2
N
2) HF/anisole
N
N
S
HN
N
HN
N
R4
N
O
NH
R2
N
R1
R1
N
N
NH
HN
N
R3
N
R4
R3
R4
TPI-1344
TPI-1347
N
R1
R3
TPI-1346
Scheme 1. Parallel synthesis of pentaamine and bis-heterocyclic libraries.
it will be elaborated, different pentaamines, and bis-heterocyclic
compounds having hydrophobic groups at R1, R2, R3 and R4 gave
interesting activities (Scheme 1).
Along with the synthesis of the mixture based libraries, 120 dif-
ferent individual compounds were synthesized for each library.
These individual compounds served as controls to determine
whether the individual building blocks used at each of the variable
positions could be successfully incorporated into the synthesis of
the corresponding libraries. The individual building blocks were
varied while the other three positions remained fixed (Scheme 2).
In vitro evaluation of antimycobacterial activity: (description of
TAACF assays)
Using the tea-bag technology and following the strategy out-
lined in Scheme 1,15 we developed an efficient approach for the
solid phase synthesis of different pentaamines and pyrrolidine
bis-heterocyclic libraries from resin-bound proline-containing
acylated tetrapeptides. Starting from resin-bound amino acids
(diversity R1), Boc-proline was coupled using standard solid
phase synthesis (SPPS) coupling reagents,16 followed by Boc
deprotection and subsequent coupling of two Boc-amino acids
(diversities R2 and R3). The N-terminal Boc was cleaved and
the generated primary amine was N-acylated with different
commercially available carboxylic acids (diversity R4). The gener-
ated resin-bound N-acylated tetrapeptide was exhaustively re-
duced using borane–THF.16 Typical reaction conditions for the
solid phase reduction of polyamides consists of the treatment
of resin-bound peptides with BH3–THF at 65 °C for 72 h.17 The
generated resin-bound borane-amine complexes are then dispro-
portionate following overnight treatment with neat piperidine at
65 °C. The reduction is free of racemization. Our approach in-
volved the use of proline as a spacer, which, following the
exhaustive reduction of the amide groups, yielded resin-bound
pentaamine containing two pairs of secondary amines separated
by a pyrrolidine ring. One set of compounds were cleaved to af-
ford the corresponding pentaamine library TPI-1343. Applying
the concept of ‘libraries from libraries’,18 extra sets of tea-bags
were prepared, where the resulting pairs of secondary amines
were separately treated with different bifunctional reagents such
as, thiocarbonyldiimidazole, cyanogen bromide and oxalyldiimi-
dazole to afford following cleavage of the solid support the cor-
responding libraries, namely pyrrolidine bis-cyclic thiourea TPI-
1347, pyrrolidine- bis-cyclic guanidines TPI-1346, and pyrroli-
dine bis-diketopiperazine TPI-1344, respectively. An extra set of
diketopiperazine was prepared and treated prior to cleavage of
the solid support with BH3-THF to afford following reduction
of the oxamide and cleavage of the resin the corresponding pyr-
rolidine bis-piperazine TPI-1345. We used the approach de-
scribed in Scheme 1, for the preparation of 5 positional scan
libraries.12,19 Twenty six different amino acids were selected
for R1, R2, and R3, and 42 carboxylic acids for R4 (Table 1) to pre-
pare in parallel, a pentaamine library and 4 different mixture
based pyrrolidine bis-heterocycles each containing 738,192 indi-
vidual compounds in positional scanning format.
1. Primary screening is conducted at 6.25 lg/mL (or molar equiv-
alent of highest molecular weight compound in a series of cong-
eners) against M. tuberculosis H37Rv (ATCC 27294) in BACTEC
12B medium using a broth microdilution assay. The Microplate
Alamar Blue Assay (MABA).20 Compounds exhibiting fluores-
cence are tested in the BACTEC 460 radiometric system. Com-
pounds affecting <90% inhibition in the primary screen (i.e.,
MIC >6.25 lg/mL) are not generally evaluated further.
2. Compounds demonstrating at least 90% inhibition in the pri-
mary screen are retested at lower concentrations against M.
tuberculosis H37Rv to determine the actual minimum inhibitory
concentration (MIC) using MABA. The MIC is defined as the low-
est concentration effecting a reduction in fluorescence of 90%
relative to controls.
3. Concurrent with the determination of MICs, compounds are
tested for cytotoxicity (IC50) in VERO cells at concentra-
tions 6 62.5 g/mL or l0 times the MIC for M. tuberculosis
l
H37Rv (solubility in media permitting). After 72 h exposure, via-
bility is assessed on the basis of cellular conversion of MTT into
a formazan product using the Promega CellTiter 96 non-radio-
active cell proliferation assay.
Screening results: Driven by the reported antitubercular activi-
ties of polyamines, 120 individual chiral pentaamines (TPI-1343)
(Scheme 1) were evaluated in a primary screen against M. tubercu-
losis (Table 2). The individual pentaamines were screened for%
inhibition against M. tuberculosis strain H37Rv at 6.25 lg/mL. Com-
pounds are considered active with inhibition >90%. Eleven com-
pounds demonstrated higher than 90% inhibition in the primary
screen and were tested at lower concentrations to determine the
minimum inhibitory concentration (MIC) by serial dilution. As
shown in Table 2, pentaamines substituted with different hydro-
phobic groups provided high inhibition at MIC values ranging from
3 to 6 lg/mL.

A. Nefzi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5169–5175
5171
Table 1
Building blocks used to synthesize the pentaamine and bis-heterocyclic libraries and resulting functionalities at the four positions of diversity
Building block used forR1, R2 and R3
Functionality
Building block used for R4
Functionality
Boc-
Boc-
L
-Ala
S-Methyl
S-Benzyl
1-Phenyl-1-cyclopropanecarboxylic acid
2-Phenylbutyric acid
(1-Phenyl-cyclopropyl)-methyl
2-Phenylbutyl
L-Phe
Boc-Gly
Hydrogen
3-Phenylbutyric acid
3-Phenylbutyl
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
Boc-
L
L
L
L
L
L
-Ile
-Leu
-Ser(Bzl)
-Thr(Bzl)
-Val
-Tyr(BrZ)
-Ala
-Phe
-Ile
S-2-Butyl
S-Isobutyl
m-Tolylacetic acid
m-Tolylethyl
3-Fluorophenylacetic acid
3-Bromophenylacetic acid
(Alpha-alpha-alpha-trifluoro-m-tolyl) acetic acid
p-Toly lacetic acid
2-(3-Fluoro-phenyl)-ethyl
2-(3-Bromo-phenyl)-ethyl
2-(3-Trifluoromethyl-phenyl)-ethyl
p-Tolylethyl
2-(4-Fluoro-phenyl)-ethyl
2-(3-Methoxy-phenyl)-ethyl
2-(4-Bromo-phenyl)-ethyl
2-(4-Methoxy-phenyl)-ethyl
2-(4-Ethoxy-phenyl)-ethyl
2-(4-Isobutyl-phenyl)-propyl
3,4-Dichlor ophe nethyl
2-(3,5-Bis-trifluoromethyl-phenyl)-ethyl
3-(3,4-Dimethoxy-phenyl)-propyl
Phenethyl
3,4,5-Trim ethoxy-benzyl
Butyl
R-Hydroxymethyl
(R,R)-1-Hydroxyethyl
S-Isopropyl
S-4-Hydroxybenzyl
R-Methyl
R-Benzyl
R-2-Butyl
R-Isobutyl
S-Hydroxymethyl
(S,S)-1-Hydroxyethyl
R-Isopropyl
R-4-Hydroxybenzyl
S-Phenyl
S-Propyl
R-Propyl
S-Butyl
R-Butyl
4-Fluorophenylacetic acid
3-Methoxyphenylacetic acid
4-Bromophenylacetic acid
4-Methoxyphenylacetic acid
4-Ethoxyphenylacetic acid
4-Isobutyl-alpha-methylphenylacetic acid
3,4-Dichlorophenylacetic acid
3,5-Bis(trifluoromethyl)-phenylacetic acid
3-(3,4-Dimethoxyphenyl)-propionic acid
Phenylacetic acid
3,4,5-Trimethoxybenzoic acid
Butyric acid
Heptanoic acid
Isobutyric acid
2-Methylbutyric acid
D
D
D
D
D
D
D
D
-Leu
-Ser(Bzl)
-Thr(Bzl)
-Val
-Tyr(BrZ)
L
-Phenylglycine
-Norvaline
L
D
-Norvaline
-Norleuc ne
-Norleucine
-Naphthylalanine
-Naphthylalanine
-Cyclohexylalanine
-Cyclohexylalanine
L
Heptyl
Isobutyl
2-Methylbutyl
3-Methylbutyl
3-Methylpentyl
4-Methylpentyl
D
L
S-2-Naphthylmethyl
R-2-Naphthylmethyl
S-Cyclohexyl
R-Cyclohexyl
D
Isovaleric acid
3-Methyvaleric acid
4-Methyvaleric acid
L
D
p-Toluic acid
4-Methyl-benzyl
Cyclopentanecarboxylic acid
Cyclohexanecarboxylic acid
Cyclohexylacetic acid
Cyclopently-methyl
Cyclohexyl-methyl
Cyclohexyl-ethyl
Cyclohexanebutyric acid
Cycloheptanecarboxylic acid
2-Methylcyclopropanecarboxylic acid
Cyclobutanecarboxylic Acid
3-Cyclopentylpropionic acid
Cyclohexanepropionic acid
4-Methyl-1-cyclohexanecarboxylic acid
4-tert-Butyl-cyclohexanecarboxylic acid
4-Biphenylacetic acid
Cyclohexyl-butyl
Cycloheptyl-methyl
(2-Methyl-cyclopropyl)-methyl
Cyclobutyl-methyl
3-Cyclopentyl-propyl
Cyclohexyl-propyl
4-Methyl-1-cyclohexyl-m ethyl
4-tert-Butyl-cyclohexyl-methyl
2-Biphenyl-4-yl-ethyl
Adamantan-1-yl-m ethyl
2-Adamantan-1-yl-ethyl
2-Bicyclo[2.2.1 ]hept-2-yl-ethyl
1-Adamantanecarboxylic acid
1-Adamantaneacetic acid
2-Norbornaneacetic acid
Encouraged by these results, we tested different bis-heterocy-
clic compounds as functionalized and constrained pentaamines
analogs, while we maintained the same R groups. One hundred
and twenty individual pyrrolidine bis-cyclic thioureas (TPI-1347),
R2
X = linearpolyamine, TPI-1343
X = S, TPI-1347
X
N
R4
N
X
X = NH, TPI-1346
X = -COCO-, TPI-1344
X = -CH2CH2-, TPI-1345
N
N
HN
R3
R1
Parallel synthesis of 120 individaul compounds for each scaffold:
(26 R1+ 26 R2+ 26 R3+ 42 R4)
R2
X
N
N
X
N
N
HN
X
N
R4
N
X
N
X
N
N
N
X
X
X
N
N
N
N
N
N
HN
HN
HN
R3
R1
42 compounds where the R4
varies and R1 , R2 and R3 are
defined as benzyl
26 compounds where the R3
varies and R1, R2 and R4 are
defined as benzyl
26 compounds where the R2
varies and R1, R3 and R4 are
defined as benzyl
26 compounds where the R1
varies and R2, R3 and R4 are
defined as benzyl
Scheme 2. Synthesis of individual controls.

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A. Nefzi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5169–5175
Table 2
MIC of chiral pentaamines against Mycobacterium tuberculosis
% Inhibition
MIC (l
g/mL)
Vero cell IC50 (lg mL)
H
N
N
99
3.13
1.1
H
N
N
N
H2
N
N
H
TPI-1343-239
H
N
N
H
91
98
3.13
6.25
1.03
4.23
H2N
N
H
TPI-1343-240
H
N
N
H
H2
N
N
H
TPI-1343-144
N
N
N
HN
N
99
6.25
1.39
TPI-1345-235
99 % Inhibition, MIC = 3.9
µ
g/mL,
Vero Cell: IC50 = 0.82µg/mL, SI = 0.21
H
N
N
99
97
92
90
96
94
99
6.25
6.25
6.25
6.25
>6.25
>6.25
n.d.
n.d.
n.d.
n.d.
1.05
n.d.
n.d.
n.d.
H
N
H2
N
N
H
TPI-1343-238
H
N
N
H
N
N
N
H2
H2
H2
N
N
H
TPI-1343-230
H
N
N
H
N
N
H
TPI-1343-233
H
N
N
H
N
N
H
TPI-1343-228
H
N
N
H
N
N
N
H2
N
N
H
TPI-1343-219
H
N
N
H
H2
N
N
H
TPI-1343-237
H
N
N
H
H2
N
N
H
TPI-1343-234
120 individual pyrrolidine bis-cyclic guanidines (TPI-1346, Scheme
3), 120 individual pyrrolidine bis-cyclic piperazines (TPI-1345),
and 120 individual pyrrolidine bis-cyclic diketopiperazines (TPI-
1344) were evaluated in a primary screen against M. tuberculosis.
These compounds were prepared according to the strategy de-
scribed in Scheme 3. They were all derived from the same penta-
amines, thus providing a double diversity of compounds, namely
a diversity of scaffolds bearing the same substituents, and a diver-

A. Nefzi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5169–5175
5173
S
NH
N
N
N
N
S
N
NH
N
N
HN
HN
N
HN
N
N
N
HN
H
N
N
N
H
H2N
N
TPI-1347-238
TPI-1346-238
TPI-1345-238
TPI-1343-238
µ
µ
µ
MIC:6.25 g/ml
% Inhibition: 98
MIC: 3.9 g/ml
MIC: 3.9 g/ml
µ
MIC: 3.13 g/ml
% Inhibition: 91
% Inhibition: 99
% Inhibition: 99
Scheme 3.
Table 3
MIC of bis-cyclic guanidines and bis-cyclic thioureas against Mycobacterium tuberculosis
NH
N
NH
N
NH
N
N
NH
N
N
N
N
HN
HN
HN
HN
NH
NH
NH
NH
N
N
N
N
N
N
N
N
Cl
Cl
g/mL,
TPI-1346-230
92 % Inhibition, MIC = 3.9
TPI-1346-213
TPI-1346-221
TPI-1346-226
µ
g/mL,
95 % Inhibition, MIC = 3.9
µ
90 % Inhibition, MIC = 7.8
µ
g/mL,
93 % Inhibition, MIC = 7.8
µ
g/mL,
Vero Cell: IC50 = 39.48
µ
g/mL, SI = 10.1
Vero Cell: IC50 = 24.48
µg/mL, SI = 6.3
Vero Cell: IC50 = 36.77
µ
g/mL, SI = 4.7
Vero Cell: IC50 = 33.54
µ
g/mL, SI = 4.3
NH
NH
N
N
NH
N
N
NH
N
N
N
HN
N
HN
HN
HN
NH
NH
N
NH
N
NH
N
N
N
N
N
N
TPI-1346-223
TPI-1346 -224
93 % Inhibition, MIC = 7.8
TPI-1346-239
91 % Inhibition, MIC = 3.9
TPI-1346-229
91 % Inhibition, MIC = 3.9µg/mL,
91 % Inhibition, MIC = 7.8
µ
g/mL,
µ
g/mL,
µ
g/mL,
Vero Cell: IC50 = 32.28
µg/mL, SI = 4.1
Vero Cell: IC50 = 31.0
µ
g/mL, SI = 3.97
Vero Cell: IC50 = 14.46 g/mL, SI = 3.7
µ
Vero Cell: IC50 = 14.34
µ
g/mL, SI = 3.7
NH
N
N
NH
NH
NH
N
N
N
N
HN
HN
N
HN
N
HN
NH
N
NH
N
NH
NH
N
N
N
N
N
N
TPI-1346-228
TPI-1346-227
TPI-1346-235
TPI-1346-240
94 % Inhibition, MIC = 7.8
Vero Cell: IC50 = 15.46 g/mL, SI = 1.98
94 % Inhibition, MIC = 3.9
µ
g/mL,
91 % Inhibition, MIC = 7.8
µ
g/mL,
91 % Inhibition, MIC = 7.8
µ
g/mL,
µg/mL,
Vero Cell: IC50 = 13.93
µ
g/mL, SI = 3.6
Vero Cell: IC50 = 26.49
µ
g/mL, SI = 3.4
Vero Cell: IC50 = 22.0µg/mL, SI = 2.8
µ
NH
N
N
S
S
N
N
HN
HN
HN
N
S
N
NH
S
N
N
N
N
N
N
TPI-1347-127
90 % Inhibition, MIC = 7.8
Vero Cell: IC50 = 3.41 g/mL, SI=44.0
TPI-1346-236
TPI-1347-231
97 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 8.47 g/mL, SI=2.2
µ
g/mL,
94 % Inhibition, MIC = 7.8µg/mL,
µ
g/mL,
µ
Vero Cell: IC50 = 15.2µg/mL, SI=1.95
µ
sity of R groups around each scaffold. In a first assay, thirty nine
bis-cyclic guanidines, six bis-cyclic thioureas and sixty individual
bis-cyclic piperazines demonstrated greater than 90% inhibition
against strain H37Rv at less than 6.25 lg/mL. Identified active
compounds were tested in a secondary assay. The results for the
times the MIC for M. tuberculosis H37Rv. The selectivity index is de-
fined as the ratio of the measured IC50 in VERO cells to the MIC. As
shown in Table 3, following the secondary screening, thirteen bis-
cyclic guanidines and only 2 bis-cyclic thioureas demonstrated
higher than 90% inhibition with MIC values ranging from 3.9 to
secondary screening of the active compounds showed inhibitions
7.8
most interesting results with 92% inhibition at 3.9
a desired selectivity index SI = 10.18 with a high cytotoxicity value
in vero cell (IC50 = 39.48 g/mL).
The pyrrolidine bis-cyclic piperazines demonstrated the highest
inhibition among all scaffolds. Following the secondary screening,
fifty compounds provided higher than 97% inhibition at low MIC
l
g/mL. The bis-cyclic guanidine TPI-1346-230 showed the
greater then 97% against strain H37Rv at less than 4
l
g/mL for some
lg/mL, having
of these compounds. Concurrent with the determination of MICs,
the individual pyrrolidine bis-cyclic thioureas (TPI-1347), individ-
ual pyrrolidine bis-cyclic guanidines (TPI-1346) and individual
pyrrolidine bis-cyclic piperazines (TPI-1345) were tested for cyto-
l
toxicity (IC50) in VERO cells at concentrations 6 62.5 lg/mL or l0

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A. Nefzi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5169–5175
Table 4
MIC of bis-cyclic piperazines against Mycobacterium tuberculosis
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
HN
HN
HN
HN
HN
N
N
N
N
N
TPI-1345-236
97 % Inhibition, MIC = 2
TPI-1345-171
100 % Inhibition, MIC = 2
Vero Cell: IC50 = 0.99 g/mL, SI = 0.50
TPI-1345-229
TP I-1345- 234
99 % Inhibition, MIC= 2
Vero Cell: IC50= 0.94 g/mL, SI= 0.47
TPI-1345-238
97% Inhibition, MIC= 2
Vero Cell: IC50= 0.97 g/mL, SI= 0.49
µ
g/mL,
µ
g/mL,
99 % Inhibition, MIC = 2
µ
g/mL,
µg/mL,
µ
g/mL ,
Vero Cell: IC50 = 1.15
µ
g/mL, SI = 0.58
µ
Vero Cell: IC50 = 1.0µg/mL, SI = 0.50
µ
µ
N
N
N
N
N
N
N
N
N
N
N
OH
N
N
N
N
N
HN
HN
HN
HN
HN
HN
HN
N
N
N
N
CF
3
Br
OH
TPI-1345-205
TPI-1345-224
TPI-1345-204
99 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 1.12 g/mL, SI = 0.29
TP I-1345- 200
99 % In hibi tion, MIC= 3.9
Vero Cell: IC50= 1.08 g/mL, SI= 0.28
TPI-1345-206
99 % Inhibition, MIC = 3.9µg/mL,
Vero Cell: IC50 = 1.12µg/mL, SI = 0.29
99 % Inhibition, MIC = 2
µ
g/mL
µg/mL,
µ
g/mL,
99 % Inhibition, MIC= 3.9µg/mL,
Vero Cell: IC50 = 0.89
µ
g/mL, SI = 0.45
µ
µ
Vero Cell: IC50= 1.11
µ
g/mL, SI= 0.28
N
N
N
N
N
N
HN
N
N
N
N
N
N
N
N
N
HN
HN
N
Cl
Cl
N
N
N
HN
N
F
TPI-1345-150
TPI-1345-202
99 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 1.05 g/mL, SI = 0.27
TPI-1345-207
TPI-1345-170
TPI-1345-213
100 % Inhibition, MIC = 3.9µg/mL,
µg/mL,
98 % Inhibition, MIC = 3.9µg/mL,
Vero Cell: IC50 = 1.05µg/mL, S I= 0.27
100 % Inhibition, MIC = 3.9µg/mL,
99 % Inhibition, MIC = 3.9µg/mL,
Vero Cell: IC50 = 1.0
µ
g/mL, SI = 0.26
µ
Vero Cell: IC50 = 1.06
µ
g/mL, SI = 0.27
Vero Cell: IC50 = 1.07
µ
g/mL, SI = 0.27
N
N
N
N
N
N
N
N
N
N
N
N
N
HN
HN
N
HN
N
N
N
N
N
HN
N
O
TPI-1345-211
TPI-1345-167
100 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 1.0 g/mL, SI = 0.26
TPI-1345-219
99 % Inhibition, MIC = 3.9
Vero Cell: IC50= 1.03 g/mL, SI = 0.26
TPI-1345-223
TPI-1345-169
98 % Inhibition, MIC =3 .9µg/mL,
µ
g/mL,
µ
g/mL,
96 % Inhibition, MIC = 3.9µg/mL,
100 % Inhibition, MIC = 3.9µg/mL,
Vero Cell: IC50 = 1.03µg/mL, SI = 0.26
µ
µ
Vero Cell: IC50 = 1.0
µg/mL, SI = 0.26
Vero Cell: IC50 = 1.0µg/mL, SI = 0.26
N
N
N
N
N
N
N
N
N
N
N
N
N
HN
N
N
N
N
HN
HN
HN
HN
N
N
N
TPI-1345-151
100 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.99 g/mL, SI = 0.25
TPI-1345-237
97 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 1.0 g/mL, SI = 0.26
TPI-1345-198
TPI-1345-172
98 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.92 g/mL, SI = 0.24
TPI-1345-199
99 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.94 g/mL, SI = 0.24
µ
g/mL,
µ
g/mL,
98 % Inhibition, MIC = 3.9µg/mL,
µ
g/mL,
µ
g/mL,
µ
µ
Vero Cell: IC50 = 0.97µg/mL, SI = 0.25
µ
µ
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
HN
HN
HN
HN
HN
N
N
N
N
N
TPI-1345-225
TPI-1345-201
TPI-1345-131
TPI-1345-197
99 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.82 g/mL, SI = 0.21
TPI-1345-240
98 % Inhibition, MIC = 3.9
µ
g/mL,
99 % Inhibition, MIC = 3.9µg/mL,
99 % Inhibition, MIC = 3.9
µ
g/mL,
µ
g/mL,
97 % Inhibition, MIC = 3.9µg/mL,
Vero Cell: IC50 = 0.88µg/mL, SI = 0.23
Vero Cell: IC50 = 0.87µg/mL, SI = 0.22
Vero Cell: IC50 = 0.86µg/mL, SI = 0.22
µ
Vero Cell: IC50 = 0.87
µ
g/mL, SI = 0.22
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
HN
HN
HN
HN
HN
N
N
N
N
F
TPI-1345-203
98 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.8 g/mL, SI=0.21
TPI-1345-235
TPI-1345-228
99 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.62 g/mL, SI = 0.16
TPI-1345-221
97 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.7 g/mL, SI = 0.18
TPI-1345-227
100 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.71 g/mL, SI=0.18
µ
g/mL,
99 % Inhibition, MIC = 3.9µg/mL,
µ
g/mL,
µ
g/mL,
µg/mL,
µ
Vero Cell: IC50 = 0.82µg/mL, SI = 0.21
µ
µ
µ

A. Nefzi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5169–5175
5175
Table 4 (continued)
N
N
N
N
N
N
N
N
N
HN
HN
HN
N
N
N
O
O
TPI-1345-210
TPI-1345-208
99 % Inhibition, MIC = 3.9
Vero Cell: IC50 = 0.32 g/mL, SI = 0.08
TPI-1345-233
99 % Inhibition, MIC = 3.9µg/mL,
µ
g/mL,
100 % Inhibition, MIC = 3.9µg/mL,
Vero Cell: IC50 = 0.33µg/mL, SI = 0.08
µ
Vero Cell: IC50 = 0.42µg/mL, SI = 0.11
values ranging from 2 to 7
played 100% inhibition at MIC values less than 4
Interestingly, the diketopiperazine analogs (TPI-1344) were not
active. They displayed low inhibition at concentrations at MIC
l
g/mL, with eight compounds that dis-
contract with the U.S. National Institute of Allergy and Infectious
Diseases. This research was supported by NIH Grants
1P41GM081261, 1P41GM079590 and funded in part by NIH Grant
1 U54 HG003916-01. The authors also thank the Multiple Sclerosis
National Research Institute, Diabetes National Group, Alzheimer’s
and Aging Research Center, Pain Management Research Institute
of America and Osteoporosis, and Breast Cancer Research Center
for their support.
lg/mL (Table 4).
higher than 6.25 lg/ml.
Analysis of the results obtained from the screening of the chiral
polyamine and bis diazacyclic libraries indicate that all the active
identified compounds have hydrophobic moieties at all R1, R2, R3
and R4 positions. Interestingly, the four scaffolds bearing the same
hydrophobic R groups showed good activities (Scheme 3), which
strengthen the suggestion that the observed activities are mainly
due to the hydrophobicity of the substituents. It is worth noting
that the adamantyl is present in all templates, this group exists
in SQ109 [N-geranyl-N’-(2-adamantyl)ethane-1,2-diamine], a no-
vel 1,2-diamine-based EMB analog, which is in advanced clinical
trials for the development of new drug for the treatment of pul-
monary tuberculosis (TB).21
References and Notes
1. Hirst, S. I. Parasitol. Today 2000, 16, 1–3.
2. Kaiser, A.; Gotrwald, A.; Wiersch, C.; Maier, W.; Seitz, H. M. Pharmazie 2002, 57,
723.
3. World Health Organization-WHO/HTM/TB/2006-http://www.who.int/tb.
4. Long, R. Can. Med. Assoc. J. 2000, 163, 425.
5. http://www.taacf.org/.
6. Schreiber, S. L. Science 2000, 287, 1964.
7. Nicolaou, K. C.; Pfefferkorn, J. A.; Barluenga, S.; Mitchell, H. J.; Roecker, A. J.; Cao,
G.-Q. J. Am Chem Soc. 2000, 122, 9968.
H
8. Toogood, P. L. J. Med. Chem. 2002, 45, 1543.
N
N
H
9. Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555.
10. Houghten, R. A.; Wilson, D. B.; Pinilla, C. Drug Discovery Today 2000, 5, 276.
11. Dolle, R. E. J. Comb. Chem. 2002, 4, 369.
SQ109
12. Houghten, R. A.; Pinilla, C.; Appel, J. R.; Blondelle, S. E.; Dooley, C. T.; Eichler, J.;
Nefzi, A.; Ostresh, J. M. J. Med. Chem. 1999, 42, 3743.
13. Hensler, M. E.; Bernstein, G.; Nizet, V.; Nefzi, A. Bioorg. Med. Chem. Lett. 2006,
16, 5073.
14. Lee, R. E.; Protopopova, M.; Crooks, E.; Slayden, R. A.; Terrot, M.; Barry, C. E., III J.
Comb. Chem. 2003, 5, 172.
15. Houghten, R. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5131.
16. Merrifield, R. B. Science 1986, 232, 341.
17. Nefzi, A.; Ostresh, J. M.; Yu, Y.; Houghten, R. A. J. Org. Chem. 2004, 69, 3603.
18. Ostresh, J. M.; Husar, G. M.; Blondelle, S. E.; Dörner, B.; Weber, P. A.; Houghten,
R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11138.
When taken in total, the results for the polyamines and con-
strained polyamine analogs as described in Scheme 1 provided a
substantial structure–activity relationship (SAR) profile. Analogs
which are fragments of the most identified active compounds will
be synthesized and tested while keeping the same chemical nature
of the R groups.
Acknowledgments
19. Houghten, R. A.; Appel, J. R.; Blondelle, S. E.; Cuervo, J. H.; Dooley, C. T.; Pinilla,
C. Biotechniques 1992, 13, 412.
20. Collins, L. A.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
21. Jia, L.; Noker, P. E.; Coward, L.; Gorman, G. S.; Protopopova, M.; Tomaszewski, J.
E. Br. J. Pharmacol. 2006, 147, 476.
Antimycobacterial data were provided by the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility (TAACF:
http://www.taacf.org/) through
a research and development