Design, synthesis, and evaluation of novel ethambutol analogues

Article abstract of DOI:10.1016/j.bmcl.2008.01.065

Ethambutol is one of the front-line agents recommended by the World Health Organization for the treatment of tuberculosis. In an effort to develop more potent therapies to treat tuberculosis, novel unsymmetrical ethambutol analogues were successfully synt

Full text of DOI:10.1016/j.bmcl.2008.01.065

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1607–1611
Design, synthesis, and evaluation of novel ethambutol analogues
Raghunandan Yendapally and Richard E. Lee*
Department of Pharmaceutical Sciences, The University of Tennessee Health Science Center,
847 Monroe Ave Rm327, Memphis, TN 38163, USA
Received 5 December 2007; revised 14 January 2008; accepted 16 January 2008
Available online 19 January 2008
Abstract—Ethambutol is one of the front-line agents recommended by the World Health Organization for the treatment of tuber-
culosis. In an effort to develop more potent therapies to treat tuberculosis, novel unsymmetrical ethambutol analogues were success-
fully synthesized by a new route utilizing novel building blocks synthesized using Ellman’s sulfinyl chemistry. The resulting
analogues were tested for anti-tuberculosis activity yielding compounds with comparable anti-tuberculosis activity to ethambutol
and increased lipophilicity that may instill better tissue penetration and serum half-life.
Ó 2008 Elsevier Ltd. All rights reserved.
Isoniazid, rifampin, pyrazinamide, and ethambutol are
H
N
H
N
the front-line agents that are recommended by World
Health Organization (WHO) for the treatment of tuber-
culosis (TB).1 The problems with the current TB treat-
ment regimens are complex and include: a prolonged
standard course regimen of 6 months, which often re-
sults in patient non-compliance; the emergence of exten-
sively drug-resistant tuberculosis strains2 (XDRTB);
and the lack of effective drugs against the latent state.
One approach to decrease the treatment time is to im-
prove the potency of currently used anti-tuberculosis
drugs. Wilkinson and coworkers from Lederle laborato-
ries first reported the synthesis and activity of ethambu-
tol (EMB) (1) (Fig. 1) in 1961.3,4 EMB is primarily a
bacteriostatic anti-tuberculosis agent. EMB targets the
arabinosyl transferases responsible for arabinogalactan
biosynthesis, a key component of the unique mycobacte-
rial cell wall.5–7 Despite modest anti-tuberculosis activ-
ity, EMB is used in combination with other front-line
anti-tuberculosis agents mainly owing to its synergy
with the other drugs and low toxicity. EMB is a simple
diamine molecule that was synthesized by reacting 1,2-
dihaloethane with chirally pure (S)-2- amino 1-buta-
nol.3,4 Based on the early SAR study it appears that
the distance between the two nitrogens, the presence of
two hydroxyl groups, and small side chains are the key
pharmacophoric elements.8 The chirality of the molecule
is also very crucial in determining the activity, as EMB,
the (S,S) isomer is approximately 500 times more potent
HO
HO
HO
N
H
OH
N
H
OH
OH
2
1
OH
N
H
N
N
H
HO
N
H
3
4
H
N
N
H
5
Figure 1. Structures of ethambutol and its analogues.
than the (R,R) isomer.8 Recently, novel EMB analogues
2, 3, and 4 were found to be active against Mycobacte-
rium smegmatis but the corresponding anti-tuberculosis
activity was not reported for these compounds.9 Lee
et al. recently synthesized a large library of asymmetrical
1, 2 diamines using combinatorial chemistry to explore
the SAR around the diamine pharmacophore. In this
process SQ 109 (5) was identified as the most active com-
pound possessing 35-fold improved activity when com-
pared to EMB.10 SQ109 has recently advanced into
clinical trials for the treatment of tuberculosis, though
it appears that SQ109 does not have the same target
as EMB as originally intended.11
In this study we further expand the SAR of unsymmet-
rical EMB derivatives by taking advantage of new chem-
istries and building blocks that were not previously
available to synthesize asymmetric and constrained
Keywords: Tuberculosis; Anti-tuberculosis; Antibiotic.
*
Corresponding author. Tel.: +1 901 448 6018; fax: +1 901 448
6828; e-mail: relee@utmem.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.065

1608
R. Yendapally, R. E. Lee / Bioorg. Med. Chem. Lett. 18 (2008) 1607–1611
analogues. Accordingly, novel EMB analogues were
synthesized and tested with an aim not only to improve
the anti-tuberculosis activity but also with a view toward
improving the pharmacokinetic profile of the emerging
compounds. Initially, EMB hydrobromide salt 6, and
its symmetrical analogues 7,12,13 and 814 were synthe-
sized by reacting amino alcohols with 1, 2-dihaloe-
thanes.3 These compounds were synthesized as
standards and the activities of these compounds were
compared with the novel unsymmetrical derivatives de-
scribed below.
To evaluate the structural importance of the side chains
and to explore non-commercially available amino alco-
O
O
a, b
H
c
TBDPSO
N
TBDPSO
Cl
HO
N
OH
N
NH2
H
H
R
10
9
11 a-d
11a: R = dimethyl
11b: R = cyclopentyl
11c: R = hydroxyl methyl
11d: R = ( )-phenyl
S
H
N
d
HO
. 2HCl
OH
NH
12 a-d
12a: R = dimethyl
R
12b: R = cyclopentyl
12c: R = hydroxyl methyl
12d: R = (S)-phenyl
Scheme 1. Reagents and conditions: (a) TBDPSiCl, imidazole, CH2Cl2, rt, 16 h, 97%; (b) chloroacetylchloride, DIPEA, CH2Cl2, rt, 16 h, 51%; (c)
amino alcohols, DIPEA, DMF, 70 °C, 14 h, 63–90%; (d) i—LiAlH4, THF, reflux, 16 h; ii—1.25 M Hydrogen chloride–methanol solution, rt, 1 h, 11–
45% yields.
OTBS
O
S
O
S
HCl.
H2N
a
c
b
OH
OTBS
N
N
H
13
14
15
O
H
H
d
HO
N
TBDPSO
N
.2HCl
OH
N
N
H
OH
H
16
17
Scheme 2. Reagents and conditions: (a) Allyl magnesium bromide, toluene, ꢀ78 °C, 3 h, 67%; (b) 4 M hydrogen chloride dioxan solution, rt, 1 h,
91%; (c) 10, DIPEA, DMF, 70 °C, 14 h, 87%; (d) i—LiAlH4, THF, reflux, 16 h; ii—1.25 M hydrogen chloride–methanol solution, rt, 1 h, 15%.
OTBS
O
S
O
S
a
H2N
c
b
R
OTBS
OH
N
N
R
H
20a, b
19a, b
19a = Allyl
19b = Ethyl
18
20a = Allyl
20b = Ethyl
O
H
H
N
d
e
TBDPSO
N
HO
N
H
OH
N
OH
H
R
R
21a, b
21a = Allyl
21b = Ethyl
22a, b
22a = Allyl
22b = Ethyl
H
N
HO
.2HCl
OH
N
H
R
23a = Allyl
Scheme 3. Reagents and conditions: (a) AllylMgBr, or EtMgCl, toluene, ꢀ78 °C, 3 h, 46% (19a), 48% (19b); (b) i—4 M hydrogen. chloride dioxan
solution, rt, 1 h; ii—Et3N, rt, 1 h (c) 10, DIPEA, DMF, 70 °C, 14 h, 45% (21a), 29% (21b); (d) LiAlH4, THF, reflux, 16 h, 56% (22b); (e) 1.25 M
hydrogen chloride–methanol solution, 19% yield from 21a to 23a.

R. Yendapally, R. E. Lee / Bioorg. Med. Chem. Lett. 18 (2008) 1607–1611
1609
hol building blocks, seven unsymmetrical EMB ana-
logues 12a–d, 17, 22b, and 23a were synthesized. Synthe-
sis of 12a–d was achieved by a novel synthetic route as
depicted in Scheme 1. In the first step alcohol 9 was pro-
tected using TBDPSiCl in the presence of imidazole in
DCM to afford silyl-protected intermediate in 97%
yield.15 N-acylation of this intermediate was achieved
using chloroacetylchloride in the presence of DIPEA
in DCM to give a-halo amide 10 in 51% yield.16 This
was subsequently reacted with a variety of commercially
available amino alcohols in the presence of DIPEA in
DMF at 70 °C for 14 h to afford respective amides
11a–d in 63–90% yields.10 TBDPS deprotection and
amide reduction were achieved in a single step using
an excess of LiAlH4 at reflux temperatures for 16 h to
yield free amino alcohols, which were subsequently con-
verted into hydrochloride salts 12a–d in 11–45% yields.20
The synthesis of S-amino alcohol 17 was performed as
described by Ellman and co-workers17 as depicted in
Scheme 2. (S)-tert-butanesulfinamide18 was reacted with
(tert-butyldimethylsilyloxy) acetaldehyde in the presence
of CuSO4 in DCM at room temperature for 24 h to yield
aldimine 13.19 Aldimine 13 was reacted with 1.0 M allyl
magnesium bromide diethyl ether solution in THF at
ꢀ78 °C for 3 h to give intermediate 14 in 67% yield. N
and O deprotection was achieved using HCl dioxan
solution to afford 2(S)-2-amino-pent-4-en-1-ol hydro-
chloride 15 in 91% yield.17 This was converted into 16
by reacting with intermediate 10 and subsequent reac-
Table 1. Structures of ethambutol analogues and their anti-tuberculosis activity
No.
Structures
M. tb H37Rv MIC90 lg/mL21
CLogPa
H
N
HO
6 EMB
0.8
0.11
. 2 HBr
. 2 HCl
N
OH
OH
H
H
N
HO
HO
7
3.12
6.25
1.6
1.36
N
H
H
N
8
ꢀ0.14
ꢀ0.01
0.74
. 2 HCl
N
H
OH
H
N
12a
12b
12c
HO
HO
. 2 HCl
. 2 HCl
N
H
OH
OH
H
N
1.6
N
H
H
N
HO
HO
. 2 HCl
3.12
ꢀ0.75
N
H
OH
OH
H
N
. 2 HCl
OH
N
H
12d
50
0.84
HO
NH
. 2 HCl
N
H
OH
17
25
0.16
0.51
0.56
HO
HO
NH
NH
22b
23a
3.12
6.25
N
H
OH
OH
N
H
. 2 HCl
a CLogP was calculated using the ChemDraw Ultra, version 7, software by Cambridge Soft.

1610
R. Yendapally, R. E. Lee / Bioorg. Med. Chem. Lett. 18 (2008) 1607–1611
tions were performed as described in Scheme 2 to afford
compound 17.
tions can be explored in detail. Even though none of
the molecules that were synthesized in this study im-
proved upon the activity of EMB, some of these mol-
ecules did have comparable in vitro activity. These
compounds now require further in vivo testing. If these
compounds retain their activity in vivo and have better
pharmacokinetic properties such as better absorption
and half-life than that of EMB then they can be con-
sidered as an alternative for replacement of EMB in
anti-tuberculosis drug therapy.
To evaluate the effect of b,b-disubstitution of the amino
alcohol unit for anti-tuberculosis activity, compounds
23a and 22b20 were synthesized by applying Ellman’s
sulfinyl chemistry as depicted in Scheme 3. (R)-tert-
butanesulfinamide18 was reacted with 1-{[tert-butyl(di-
methyl)silyl] oxy}acetone in the presence of Ti(OEt)4
in THF at 70 °C to give ketamine 18,19 which was sub-
sequently treated with allyl magnesium bromide and
ethyl magnesium chloride to give intermediate 19a
(46% yield) and 19b (48% yield). N and O deprotection
was achieved using HCl dioxan solution to yield 20a and
20b.17 Final products 23a and 22b were obtained by
reacting amino alcohols 20a and 20b with intermediate
10 and performing subsequent reactions as described
in Scheme 3.
Acknowledgments
We thank Robin Lee and Mitchell Lingerfelt for their
technical assistance. This work was supported by Grant
AI057836 from the National Institute Health.
To establish the structure–activity relationship (SAR) of
EMB, compounds in Table 1 were tested for their anti-
tuberculosis MIC90 activity against M. tuberculosis
H37Rv.21 Our resynthesized EMB standard (6) had an
MIC of 0.8 lg/mL. Symmetrical compounds with cyclo-
pentyl side chain (7) and dimethyl (8) side chain were
less active than that of the EMB salt (6) which is consis-
tent with previous reports.
References and notes
1. World Health Organization. http://whqlibdoc.who.int/hq/
2003/WHO_CDS_TB_2003.313_eng.pdf.
2. Shah, N. S.; Wright, A.; Bai, G.-H.; Barrera, L.; Boulah-
bal, F.; Martin-Casabona, N.; Drobniewski, F.; Gilpin,
C.; Havelkova, M.; Lepe, R.; Lumb, R.; Metchock, B.;
Portaels, F.; Rodrigues, M. F.; Rusch-Gerdes, S.; Van
Deun, A.; Vincent, V.; Laserson, K.; Wells, C.; Cegielski,
J. P. Emerging Infect. Dis. 2007, 13, 380.
Unsymmetrical compounds in which one-half of the
ethyl side chain of EMB was replaced with smaller di-
methyl (12a), cyclopentyl (12b), and hydroxy methyl
(12c) side chains, resulted in MIC values in between
EMB and the corresponding symmetrical derivatives (8
and 7). Replacement of the ethyl side chain with larger
(S) allyl (17) and (S) phenyl (12d) side chains had a large
detrimental effect on activity. Interestingly, the replace-
ment of hydrogen at the chiral carbon of the amino alco-
hol section of (17) with a methyl group (23a) resulted in
increased anti-tuberculosis activity. However, the analo-
gous substitution to the chiral center of EMB (6) re-
duced anti-tuberculosis activity (22b). The tight SAR
observed for the compounds in this study is consistent
with previous reports for ethambutol analogues in the
literature.8,9 Therefore, careful attention must be paid
when designing new potential EMB analogues. Cur-
rently we believe that modifications that also seek to im-
prove the pharmacokinetic properties of EMB rather
than simply the improve the anti-tuberculosis activity
may be a more productive approach. The CLogP values
for the compounds in this study were estimated using
ChemDraw Ultra and are reported in Table 1. Most
of the synthesized novel compounds had higher CLogP
values than that of EMB indicating that they may have
better pharmacokinetic profiles leading to an increase in
cerebrospinal fluid (CSF) penetration, serum binding
and serum half-life. EMB analogues with these proper-
ties may be more useful the treatment of CSF infections
than EMB, which has limited application due to moder-
ate penetration into the CSF.
3. Wilkinson, R. G.; Shepherd, R. G.; Thomas, J. P.;
Baughn, C. J. Am. Chem. Soc. 1961, 83, 2212.
4. Thomas, J. P.; Baughn, C. O.; Wilkinson, R. G.; Shep-
herd, R. G. Am. Rev. Respir. Dis. 1961, 83, 891.
5. Lee, R. E.; Mikusova, K.; Brennan, P. J.; Besra, G. S.
J. Am. Chem. Soc. 1995, 117, 11829.
6. Zhang, J.; Khoo, K. H.; Wu, S. W.; Chatterjee, D. J. Am.
Chem. Soc. 2007, 129, 9650.
7. Alderwick, L. J.; Seidel, M.; Sahm, H.; Besra, G. S.;
Eggeling, L. J. Biol. Chem. 2006, 281, 15653.
8. Shepherd, R. G.; Baughn, C.; Cantrall, M. L.; Goodstein,
B.; Thomas, J. P.; Wilkinson, R. G. Ann. NY. Acad. Sci.
1966, 135, 686.
9. Hausler, H.; Kawakami, R. P.; Mlaker, E.; Severn, W. B.;
Stutz, A. E. Bioorg. Med. Chem. Lett. 2001, 11, 1679.
10. Lee, R. E.; Protopopova, M.; Crooks, E.; Slayden, R. A.;
Terrot, M.; Barry, C. E., III J. Comb. Chem. 2003, 5, 172.
11. Boshoff, H. I.; Myers, T. G.; Copp, B. R.; McNeil, M. R.;
Wilson, M. A.; Barry, C. E., 3rd J. Biol. Chem. 2004, 38,
40174.
12. Cremieux, A.; Baghdadi, N.; Berthelot, P.; Debaert, M.
Ann. Microbiol. (Paris). 1983, 134A, 177.
13. Berthelot, P.; Debaert, M.; Cremieux, A.; Baghadi, N.
Farmaco, Edizione Scientifica. 1983, 38, 73.
14. Wilkinson, R. G.; Cantrall, M. B.; Shepherd, R. G.
J. Med. Pharmaceut. Chem. 1962, 5, 835.
15. Brickmann, K.; Yuan, Z.; Sethson, I.; Somfai, P.; Kihl-
berg, J. Chem. Eur. J. 1999, 5, 2241.
16. Kim, K. S.; Kimball, S. D.; Misra, R. N.; Rawlins, D. B.;
Hunt, J. T.; Xiao, H.-Y.; Lu, S.; Qian, L.; Han, W.-C.;
Shan, W.; Mitt, T.; Cai, Z.-W.; Poss, M. A.; Zhu, H.;
Sack, J. S.; Tokarski, J. S.; Chang, C. Y.; Pavletich, N.;
Kamath, A.; Humphreys, W. G.; Marathe, P.; Bursuker,
I.; Kellar, O. K. A.; Roongta, U.; Batorsky, R.; Mulher-
on, J. G.; Bol, D.; Fairchild, C. R.; Lee, F. Y.; Webster, K.
R. J. Med. Chem. 2002, 45, 3905.
In summary we have developed a new route for the
synthesis of novel EMB analogues. In future, using this
new synthetic route many more side chain modifica-
17. Tang, T. P.; Volkman, S. K.; Ellman, J. A. J. Org. Chem.
2001, 66, 8772.

R. Yendapally, R. E. Lee / Bioorg. Med. Chem. Lett. 18 (2008) 1607–1611
1611
18. Liu, G.; Cogan, D. A.; Ellman, J. A. J. Am. Chem. Soc.
1997, 119, 9913.
19. Liu, G.; Cogan, D. A.; Owens, T. D.; Tang, T. P.; Ellman,
J. A. J. Org. Chem. 1999, 64, 1278.
pentet), 2.52–2.68 (4H, m), 3.32–3.38 (2H, m), 3.41 (1H, dd,
J = 5.85, 11.47 Hz), 3.51 (1H, dd, J = 4.63, 11.47 Hz); ESI-
MS: 219.2 (M+1); ½aꢁD26:3=+10.0 (c = 1%, MeOH). Anal.
Calcd for C11H26N2O2: C, 60.51; H, 12.0; N, 12.83. Found:
C, 60.58; H, 12.1; N, 12.74. Compound: 23a: 1H NMR
(500 MHz, D2O): 0.87 (3H, t, J = 7.56 Hz), 1.21 (3H, s),
1.54–1.71 (2H, m), 2.37 (2H, d, J = 7.56 Hz), 3.17 (1H,
sextet), 3.3-3.4 (4H, m), 3.5–3.62 (2H, m), 3.66 (1H, dd,
J = 5.37, 13.18 Hz), 3.78 (1H, dd, J = 3.17, 12.93 Hz), 5.16-
5.24 (2H, m), 5.68-5.78 (1H, m); ESI-MS: 217.2 (M+1).
21. MIC values were determined against M. tuberculosis
H37Rv by the microbroth dilution method. A broth
culture of M. tuberculosis was grown in Middlebrook 7H9
medium with 10% ADC supplement to an OD600 of 0.4–
0.6. The culture was diluted with 7H9 medium to an
OD600 of 0.01, and 100 lL was added to a microtiter plate
containing twofold serial dilutions of the ethambutol
analogues for a final volume of 200 lL. The plates were
incubated at 37 °C for 7 days. The MIC90 was determined
by visual inspection and defined as the concentration that
inhibited 90% of growth.
20. Analytical data for a representative compounds Com-
pound 12a: 1H NMR (500 MHz, D2O): 0.87 (3H, t,
J = 7.56 Hz), 1.22 (6H, s), 1.54–1.72 (2H, m), 3.17 (1H,
sextet), 3.26–3.32 (2H, m), 3.34–3.42 (2H, m), 3.51 (2H, s),
3.66 (1H, dd, J = 5.12, 12.93 Hz), 3.78 (1H, dd, J = 3.17,
25:7
12.93 Hz); ESI-MS: 205.2 (M+1). ½aꢁD +5.7 (c = 1.25 %,
MeOH). Anal. Calcd for C10H26Cl2N2O2: C, 43.32; H,
9.45; N, 10.1. Found: C, 43.23; H, 9.35; N, 9.52. Com-
pound: 12b: 1H NMR (500 MHz, D2O): 0.87 (3H, t,
J = 7.32 Hz), 1.54–1.82 (10H, m), 3.18 (1H, sextet), 3.28–
3.34 (2H, m), 3.36–3.42 (2H, m), 3.55 (2H, s), 3.66 (1H, dd,
J = 5.37, 13.18 Hz), 3.79 (1H, dd, J = 3.17, 12.93 Hz); ESI-
26:4
MS: 231.2 (M+1). ½aꢁD +3.1 (c = 1 %, MeOH). Anal.
Calcd for C12H28Cl2N2O2: C, 47.52; H, 9.31; N, 9.24.
Found: C, 47.51; H, 9.23; N, 9.1. Compound: 22b: 1H
NMR (500 MHz, D2O): 0.74 (3H, t, J = 7.56 Hz), 0.79 (3H,
t, J = 7.56 Hz), 0.88 (3H, s), 1.36–1.44 (4H, m), 2.48 (1H,