Novel Heteroarotinoids as Potential Antagonists of Mycobacterium bovis BCG

Article abstract of DOI:10.1021/jm0303453

A series of 15 heteroarotinoids has been prepared and evaluated for activity against Mycobacterium bovis BCG with the thiourea-containing isoxyl (7) (0.5 μg/mL) as the standard. 2,2,4-Trimethyl-2H-chromen-7-yl 4-(methoxycarbonyl)benzoate (8) displayed the most significant activity (2.0-4. 0 μg/mL) in terms of the lowest concentration (μg/mL) (MIC, minimum inhibitory concentration) required to produce a 99% reduction in the number of colonies on a plate as compared to that system free of the agent at the same dilution of the culture suspension. Ethyl 4-{[N-(2,2,4,4-tetramethylchroman-6-yl)thiocarbamoyl]amino}benzoate (9) and [(1E,3Z,5E)-1-aza-4-methyl-6-(1,2,2,4-tetramethyl(1,2-dihydroquinolyl))hexa-1,3, 5-trienyl]amino}aminomethane-1-thione (10) exhibited activity at 5.0-10.0 and 10.0-20.0 μg/mL, respectively, while the other examples had MIC values of 20 μg/mL or greater. The inhibitory ability of 8 may occur via the inhibition of mycolic acid synthesis in a like manner as found with 7, but this requires further study. The heteroarotinoids are the first examples to exhibit inhibitory ability against the growth of Mycobacterium bovis BCB.

Full text of DOI:10.1021/jm0303453

1008
J . Med. Chem. 2004, 47, 1008-1017
Novel Heter oa r otin oid s a s P oten tia l An ta gon ists of Mycoba cter iu m bovis BCG
Chad W. Brown,† Shengquan Liu,† J ozef Klucik,† K. Darrell Berlin,*,† Patrick J . Brennan,*,‡
Devinder Kaur,‡ and Doris M. Benbrook§
Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-3071, Department of Microbiology,
Colorado State University, Fort Collins, Colorado 80523-1677, and Department of Obstetrics & Gynecology and Department of
Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
Received J uly 16, 2003
A series of 15 heteroarotinoids has been prepared and evaluated for activity against
Mycobacterium bovis BCG with the thiourea-containing isoxyl (7) (0.5 µg/mL) as the standard.
2,2,4-Trimethyl-2H-chromen-7-yl 4-(methoxycarbonyl)benzoate (8) displayed the most signifi-
cant activity (2.0-4.0 µg/mL) in terms of the lowest concentration (µg/mL) (MIC, minimum
inhibitory concentration) required to produce a 99% reduction in the number of colonies on a
plate as compared to that system free of the agent at the same dilution of the culture suspen-
sion. Ethyl 4-{[N-(2,2,4,4-tetramethylchroman-6-yl)thiocarbamoyl]amino}benzoate (9) and
{[(1E,3Z,5E)-1-aza-4-methyl-6-(1,2,2,4-tetramethyl(1,2-dihydroquinolyl))hexa-1,3,5-trienyl]-
amino}aminomethane-1-thione (10) exhibited activity at 5.0-10.0 and 10.0-20.0 µg/mL,
respectively, while the other examples had MIC values of 20 µg/mL or greater. The inhibitory
ability of 8 may occur via the inhibition of mycolic acid synthesis in a like manner as found
with 7, but this requires further study. The heteroarotinoids are the first examples to exhibit
inhibitory ability against the growth of Mycobacterium bovis BCB.
In tr od u ction
continues to claim more lives than any other single
infectious agent.4 Its pathogenicity is believed to arise
from an ability to survive in host cells by colonizing
macrophages and remaining quiescent for long periods
of time, only to become active decades later.5 Ap-
proximately one-third of the world’s population is
infected with Mtb, 10% of which are predicted to develop
the disease over the course of their lives.6 One current
treatment for active TB is a four-drug regimen com-
prised of isoniazid, rifampin, pyrazinamide, and etham-
butol for a period of at least 6 months.7 A failure of
patients to complete the therapy has led to the emer-
gence of multidrug-resistant (MDR) tuberculosis. The
increasing number of cases of MDR tuberculosis has
become such a public health threat that the World
Health Organization (WHO) has declared TB a global
public health emergency.8 Consequently, there is an
urgent need to create high efficacy or rapid-acting
agents to treat Mtb.
The biology-chemistry of retinoids is an extremely
active area, especially with new synthetic examples for
the treatment of malignancies1 and dermatological
disorders.2 Heteroarotinoids (1) are structurally related
to trans-retinoic acid (t-RA, 2), but the former are
generally much less toxic.3 Tazarotene (3) is an example
of a heteroarotinoid that is currently in clinical trials
for the treatment of psoriasis.2 To the best of our
knowledge, there are no reports of the utility of hetero-
arotinoids as chemotherapeutic agents to treat any
strains of mycobacteria.
Mycobacterium tuberculosis (M. tuberculosis, or Mtb),
a human pathogen causing tuberculosis (TB), is respon-
sible for the death of millions of people each year and
One approach is the development of agents that will
successfully inhibit the actions of the enzyme dihydro-
folate reductase (DHFR). DHFR is essential for folate
metabolism in both eukaryotic and prokaryotic cells.9
It is important in the treatment of many types of
infections10 and in the treatment of patients with HIV
and Mtb infections.11 Agents such as methotrexate
[MTX (4), an antiinflammatory and immunosuppressive
agent],12 trimethoprim (TMP, 5),5 and Br-WR99210 [6,5
a bromine analogue of triazine (WR99210)] are impor-
tant as DHFR inhibitors. A structural comparison study
of DHFRs from human tissue and M. tuberculosis
performed by Hol and co-workers5 provided several
useful observations. Through a sequence alignments of
the amino acids in the DHFRs of human and Mtb, Hol
* Corresponding authors. For biology (P.J .B.): e-mail, Patrick J .
Brennan@colostate.edu. For chemistry (K.D.B.): e-mail, kdb@
okstate.edu.
† Oklahoma State University.
‡ Colorado State University.
§ University of Oklahoma Health Sciences Center.
10.1021/jm0303453 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/09/2004

Heteroarotinoids as Antagonists of M. bovis BCG
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1009
duction of the nitro groups in a mixture of these isomers
was achieved in highest conversions with iron in acetic
acid and absolute ethanol and provided 29a and 29b,
which were easily separated by chromatography. In fact,
later experiments revealed that the best returns of 29a
and 29b were realized by subjecting the crude mixture
of 28a and 28b to reducing conditions and then separat-
ing 29a and 29b via immediate chromatography.
Treatment of 29a and/or 29b with the correspondingly
required isocyanate or isothiocyanate as illustrated in
Scheme 2 led to 9, 11, and 13 as well as to 19, 22, and
21, respectively. Yields of the ureas and thioureas were
good to excellent with all of the solids being purified by
recrystallization to sharp melting points. Obviously, the
urea or thiourea groups in the 6- or 8-positions in the
systems could elicit a different response in the assay.
To assess the impact of the sulfur heteroatom on
activity, as compared to that observed with the oxygen-
containing cyclohexyl ring systems 9, 11, and 13, esters
15 and 17 were examined. Although reported earlier,14
no preparations were disclosed for these latter deriva-
tives. Starting with 26,15 the condensation was straight-
forward and gave 15 and 17 under conditions similar
to those reported for 9, 11, 13, 19, 20, and 21.
A series of nitrogen-possessing analogues was inves-
tigated to ascertain the influence of this heteroatom and
also the substituent on the cyclohexyl ring unit in the
form of one less methyl group at C-4. The condensation
of aniline with acetone and a trace of iodine in an acidic
medium afforded 30, which was an oil and was used at
once to prepare the N-methyl derivative 27, also an oil
(Scheme 3). Likewise, 27 was immediately treated with
LiClO4 and freshly prepared bis(2,2,2-trichloroethyl)azo-
dicarboxylate (31) in ether. Decomposition of the inter-
mediate azide with zinc/concentrated acetic acid af-
forded 32 as a solid (51%). To avoid degradation, 32 was
treated immediately with the appropriate thioisocyan-
ates/isocyanate and gave 12, 14, and 16.
noted only a ∼26% sequence identity and indicated key
differences that might be considered in the future
development of selective inhibitors of the DHFR of Mtb.
Isoxyl (7) has demonstrated potent activity against
various Mycobacterium strains, including M. tubercu-
losis (Mtb).13 Isoxyl is also known to inhibit mycolic acid
synthesis and fatty acid synthesis in the same bacte-
ria.13 As can be noted, 7 contains a flexible thiourea
group connected to two aryl rings, which are also in 4-6.
Similar H-bonding NH groups are in 4-7, possibly
important for inhibition. We discovered that certain
heteroarotinoids with thiourea linkages exhibit strong
anticancer activity.14,15 We now report the antimyco-
bacterial activity of some heteroarotinoids with H-
bonding groups and structurally related to 7 but with
saturated rings fused to one aryl ring.
Ch em istr y
To determine if an extended side chain would induce
stronger activity in this nitrogen series, 27 was sub-
jected to formylation with POCl3/DMF and yielded 33
(Scheme 4). Lactonization of 33 in a base-catalyzed
experiment with ethyl 3,3-dimethylacrylate generated
34, which was immediately reduced with DIBAL-H in
THF at -78 °C and gave 35. Ring opening of 35 under
dilute acid conditions in 1,2-dichloroethane led to alde-
hyde 36. Treatment of 36 with thiosemicarbazone in
acetic acid-ethanol at 60 °C produced the thiocarbazone
10. Another reason for assessing 10 in the current assay
was based upon previous work with two heteroaro-
tinoids that gave thiocarbazones that exhibited retinoid
receptor activation and/or growth inhibition of ovarian
cancer cells.3,14
None of the heteroarotinoids reported herein are
commercially available. The preparation of members of
8-22 (Table 1) required the use of key starting materi-
als 23-27. Direct methods to obtain such intermediates
For comparison purposes, 8 was targeted for synthe-
sis, since it had oxygen rather than nitrogen in the
cyclohexyl ring, was void of one methyl group at C-4,
contained a shorter linking ester group, and had the
linking group at C-7. Lactone 3717 reacted with excess
methylmagnesium bromide in THF at room tempera-
ture for 3 days to give 25 in modest yield (40%) (Scheme
5). The anion of 25 condensed smoothly with the
required acid chloride shown and created ester 8.
have been generally laborious. Heterocycles 2415 and
2615 are known, while 2316 and 2517 have been partially
characterized and 27 is unknown. Structures of the
latter three materials have now been firmly established.
Direct nitration of 23 could only be accomplished in
reasonable yield at -5 °C by using a combination of
nitric acid and freshly distilled acetic anhydride (Scheme
1). A mixture of isomers 28a and 28b was isolated in
pure form as creamy white solids in yields of 43% and
26%, respectively, after rigorous chromatography. Re-
Changing the position of the heteroatom in the ring
was the impetus for preparing 18 and 20. In addition,

1010 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Brown et al.
Ta ble 1. Antibacterial Activity of Selected Heteroarotinoids against M. bovisa
a
The results are from the treatment of M. bovis (BCG) with the agents listed. See the Experimental Section for details of the assay.
b
MIC is the lowest concentration (µg/mL) of an agent resulting in a 99% reduction in the number of colonies on a plate as compared to
those on a plate free of the agent at the same dilution of the culture suspension.
Sch em e 1
Sch em e 2
to 39 (89%) proved to be the use of TiCl3 in an acidic
medium. Treatment of 39 with 4-nitrophenyl thioiso-
cyanate in THF afforded 18 under the usual conditions.
In the reaction of 39 with 4-nitrophenylbenzoyl isothio-
cyanate, all reagents and equipment had to be excep-
tionally dry to avoid severe mixtures in the product.
Although 20 was a solid and could be recrystallized, it
retained a small amount of water in the solid state, even
after extensive drying. The rationale for the additional
atom in the linking group of 20, compared to that in
18, was to give a measure of activity with a more flexible
linker.
inserting a group at C-7 would alter the degree of
rotation around the thiourea-type linking group and
might well affect the biological activity of the com-
pounds. Nitration of 2415 under conditions as illustrated
in Scheme 6 gave a mixture of the 5-nitro isomer, the
7-nitro isomer, and some dinitro-substituted compound.
Proton NMR analysis revealed a ratio of approximately
3:5:1, respectively, of three products. Repeated and very
careful flash chromatography allowed the isomer 38
(32%) to be obtained, but it was best reduced at once to
amine 39. The optimum conditions for reduction of 38

Heteroarotinoids as Antagonists of M. bovis BCG
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1011
Sch em e 3
Sch em e 4
Sch em e 5
eroarotinoids 9, 11, and 12 are structurally similar to
7, the activities of the former are reduced. In contrast,
agent 8, which is less structurally similar to 7, demon-
strated the best activity against BCG with a MIC value
of 2.0-4.0 µg/mL.
Resu lts a n d Discu ssion
In summary, 15 heteroarotinoids were evaluated for
antibacterial activity against M. bovis with isoxyl (7)
as the reference. Compound 8 displayed the best activity
with a MIC of 2.0-4.0 µg/mL. Both 9 and 10 exhibited
MIC values of 5.0-10 and 10.0-20.0 µg/mL, respec-
tively, while all other examples had values >20.0 µg/
mL. It is speculated that the antibacterial activity may
be the result of the inhibition of mycolic acid synthesis
and/or the inhibition of DHFR. The latter might result
from the cleavage of the ester group in 8, for example,
to a carboxyl anion, as certain studies conclude from
the interactions of some endogenous ligands with recep-
tor proteins,15,18,19 and a “salt bridge” could be created
via interactions with Arg32 and Arg60, as in 4.5 Another
observation5 made on the DHFR enzyme was that
inhibitors 4-6 adopted a “curved conformation” with the
nitrogen-containing rings oriented toward the binding
site in the interior of the enzyme, while the aromatic
ring was closer to the surface. The structure of 8 is
slightly shorter than that of 4 or 6 and can assume a
near linear conformation and/or a curved conformation
Biologica l Activity
The 15 heteroarotinoids listed in Table 1 were tested
for antibacterial activity against Mycobacterium bovis
(BCG) and compared to that of Isoxyl (7) by the
technique previously reported from our group.13 The
results are listed as MIC values in Table 1. The MIC
value has been defined as the lowest concentration (in
µg/mL) of drug resulting in 99% reduction in the number
of bacterial colonies on a plate compared to those on a
plate free of the drug at the same dilution of the
bacterial culture suspension.13 Isoxyl (7) demonstrated
a MIC value of 0.5 µg/mL against BCG (Table 1),
indicating potent antibacterial activity. Several of the
heteroarotinoids exhibited modest antibacterial activity,
with MIC values of 20.0-40.0 µg/mL. Compounds 9 and
10 showed promising activity with MIC values of 5.0-
10.0 and 10.0-20.0 µg/mL, respectively. Although het-
Sch em e 6

1012 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Brown et al.
viscous oil, which was subjected to flash chromatography
as judged by molecular models. Consequently, 8 may
be able to locate between the interior, hydrophobic
amino acid residues as well as extending toward the
exterior Arg32 and Arg60 residues of the ligand binding
site within the enzyme. Such a position could endow 8
with antibacterial properties. Further work is needed
to delineate a more exact role of heteroarotinoids in
terms of antibacterial chemotherapy. The compounds
herein are the first examples of heteroarotinoids to
display inhibitory ability against M. bovis.
[Et2O:hexanes (1:1)] to afford 9 (284 mg, 69%) as a flaky, white
solid: mp 102-104 °C; IR (KBr) 3351, 3289, 1714 cm-1 1H
;
NMR (DCCl3) δ 1.35 (m, 15 H), 1.85 (s, 2 H), 4.35 (q, J ) 7.16
Hz, 2 H), 6.85 (d, J ) 8.07 Hz, 1 H), 7.04 (dd, J ) 7.98, 2.07
Hz, 1 H), 7.24 (d, J ) 2.04 Hz, 1 H), 7.57 (d, J ) 8.12 Hz, 2 H),
7.81 (bs, 1 H), 8.01 (d, J ) 8.05 Hz, 2 H), 8.09 (bs, 1 H); 13C
NMR (DCCl3) ppm 14.28, 28.45, 31.06, 32.70, 48.47, 60.96,
75.09, 119.39, 123.01, 124.85, 124.92, 127.47, 128.25, 130.42,
133.32, 141.99, 152.35, 152.35, 165.85, 179.47. Anal. (C23H28
N2O3S) C, H, N, S.
-
{[(1E ,3Z,5E )-1-Aza -4-m e t h yl-6-(1,2,2,4-t e t r a m e t h yl-
(1,2-d ih yd r oq u in olyl))h exa -1,3,5-t r ien yl]a m in o}a m in o-
m eth a n e-1-th ion e (10). To thiosemicarbazide (71.31 mg, 0.76
mmol), dissolved in 4 mL of water and AcOH (1 drop), was
added dropwise a warmed (60 °C) solution of aldehyde 36 (200
mg, 0.71 mmol) dissolved in EtOH (95%, 5 mL). A precipitate
formed immediately. The reaction mixture stood for 24 h (0
°C), and then the solid was filtered off. Recrystallization
(EtOAc:diethyl ether, 1:1) of the solid afforded 10 (123 mg,
41%) as a light orange solid: mp 177-179 °C; IR (neat) 3428,
Exp er im en ta l Section
Gen er a l. Commercial reagents and solvents were used as
received unless otherwise noted. Anhydrous THF was obtained
by distillation from a purple solution of sodium and benzo-
phenone. All of the isocyanates and isothiocyanates were
commercially available and were obtained from Lancaster
Synthesis, Inc., Windham, NH. Unless otherwise indicated,
all reactions were run under dry N2, and all purification
beyond recrystallization was done by flash chromatography
(J . T. Baker flash chromatography silica gel packing, 40 µm
particle size). Melting points were determined on a Thomas-
Hoover melting point apparatus and were uncorrected. IR
spectra were obtained using a Perkin-Elmer 2000 FT-IR
spectrometer as films or KBr pellets. Unless stated otherwise,
all 1H and 13C NMR spectra were performed using DCCl3
(unless otherwise noted) as the solvent and obtained on a
Varian UNITYINOVA 400 BB NMR spectrometer operating at
399.99 and 100.01 MHz, respectively. Signals were referenced
to TMS. Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA. Although the intermediates and
final products appeared relatively stable in light, precautions
were taken to minimize exposure to any light source and to
the atmosphere. In the purified state, all products were stored
in the cold and dark without significant decomposition.
Compounds 24 and 26 were prepared by known methods.15
1
3254, 3156 cm-1; H NMR (DCCl3) δ 1.32 (s, 6 H), 2.01 (d, 3
H), 2.06 (d, 3 H), 2.84 (s, 3 H), 5.32 (d, 1 H), 6.17 (d, 1 H), 6.47
(d, 1 H), 6.72 (s, 1 H), 7.14-7.25 (m, 3 H); 7.88 (s, 1 H), 7.91
(s, 1H), 9.28 (s, 1 H), 13C NMR (DCCl3) ppm, 18.57, 27.64,
30.79, 56.63, 110.55, 121.81-155.04, 177.75. Anal. (C20H26N4S)
C, H, N.
[(4-Nitr op h en yl)a m in o][(2,2,4,4-tetr a m eth ylch r om a n -
6-yl)a m in o]m et h a n e-1-t h ion e (11). The compound was
prepared from amine 29a and 4-nitrophenyl isothiocyanate
(187 mg, 1.04 mmol). Evaporation of the solvent gave a solid,
which was subjected to flash chromatography (Et2O:hexanes,
2:1) to afford 11 (327 mg, 85%) as a light yellow solid: mp
166-168 °C; IR (KBr) 3346, 3215 cm-1 1H NMR (DCCl3) δ
;
1.35 (s, 6 H), 1.38 (s, 6 H), 1.86 (s, 2 H), 6.87 (d, J ) 7.98 Hz,
1 H), 7.04 (dd, J ) 8.01, 2.06 Hz, 1 H), 7.23 (d, J ) 2.01 Hz,
1 H), 7.74 (d, J ) 8.03 Hz, 2 H), 7.87 (bs, 1 H), 8.18 (d, J ) 8.0
Hz, 2 H) 8.35 (s, 1 H); 13C NMR (DCCl3) ppm 28.45, 31.09,
32.72, 48.38, 75.21, 119.66, 122.81, 124.42, 124.89, 127.69,
133.65, 144.03, 144.32, 152.71, 179.15. Anal. (C20H23N3O3S)
C, H, N, S.
2,2,4-Tr im eth yl-2H-ch r om en -7-yl 4-(Meth oxyca r bon -
yl)ben zoa te (8). A suspension of NaH (0.054 g, 2.14 mmol,
1.02 equiv) in dry THF (1 mL) was chilled to 0 °C, and then
25 (0.400 g, 2.10 mmol) in dry THF (3 mL) was added
dropwise. When the addition was complete (∼10 min), monom-
ethyl terephthaloyl chloride (04.64 g, 2.33 mmol, 1.1 equiv)
was added dropwise. The resulting mixture was allowed to
warm to room temperature slowly and then was stirred for
12 h. The final mixture was poured into water (15 mL)
containing 3 drops of glacial acetic acid. Two layers separated,
and the water layer was extracted (EtOAc, 4 × 15 mL). The
combined organic layer and extracts was washed with 10%
NaOH (2 × 15 mL), water (15 mL), and brine (15 mL). After
drying (Na2SO4), the solution was evaporated to a solid which
was recrystallized (HCCl3:pentane, 1:1) to afford 8 (0.270 g,
36%) as a shiny, white solid: mp 94-95 °C; IR 1740, 1731
E t h yl 4-({[(1,2,2,4-Tet r a m et h yl-1,2-d ih yd r oq u in ol-6-
yl)a m in o]th ioxom eth yl}a m in o)ben zoa te (12). To a solu-
tion of (1,2,2,4-tetramethyl-1,2-dihydroquinol-6-yl)amine (32,
150 mg, 0.74 mmol) in 4 mL of dry THF in oven-dried
equipment and cooled to -5 °C was added dropwise ethyl
4-isothiocyanatobenzoate (161 mg, 7.78 mmol, 1.05 equiv)
dissolved in dry THF (5 mL, 30 min). After the addition, the
reaction mixture was allowed to warm to room temperature
and was then stirred (24 h). The solvent was evaporated to a
solid which was recrystallized (H2CCl2:pentane, 1:2) to afford
12 (275 mg, 91%) as a pale yellow solid: mp 161-162 °C; IR
1
(KBr pellet) 3344, 3289, 1712 cm-1; H NMR (DCCl3) δ 1.34
(s, 6 H), 1.37 (t, 3 H), 1.60 (bs, 1 H), 1.95 (s, 3 H), 2,83 (s, 3 H),
4.35 (q, 2), 5.36 (s, 1 H), 6.51 (d, 1 H, J ) 8.7 Hz), 6.94 (d, 1 H,
J ) 2.4 Hz), 7.13 (q, 1 H, J ) 2.4 Hz, J ) 8.7 Hz), 7.60 (d, 2
H, J ) 8.7 Hz), 7.75 (bs, 1 H), 8.01 (d, 2 H, J ) 8.7 Hz); 13C
NMR (DCCl3) ppm 14.30, 18.53, 27.77, 30.86, 56.75, 60.92,
1
cm-1; H NMR (DCCl3) δ 1.41 (s, 6 H), 2.01 (d, 3 H, J ) 1.89
Hz), 3.96 (s, 3 H), 5.41 (m, 1 H), 6.68 (d, 1 H, J ) 2.0 Hz), 6.74
(dd, 1 H, J ) 2.01, 7.97 Hz), 7.17 (d, 1 H, 7.98 Hz) 8.16 (d, 2
H, 8.01 Hz), 8.24 (d, 2 H, J ) 8.02 Hz); 13C NMR (DCCl3) ppm
17.94, 28.15, 52.46, 76.48, 109.76, 113.25, 121.08-153.94,
164.17, 166.14. Anal. (C21H20O5) C, H.
111.04, 121.66, 123.06-143.5, 165.97, 179.92. Anal. (C23H27
N3O2S) C, H, N.
-
Gen er a l P r oced u r e for t h e P r ep a r a t ion of H et er o-
a r otin oid s 9, 11, 13, 19, 21, a n d 22 fr om Cor r esp on d in g
Am in e 29a or 29b. To a solution of amine 29a (205 mg, 1.0
mmol) or 29b (205 mg, 1.0 mmol) in dry THF (4.5 mL) at 0 °C
was added dropwise the desired isocyanate or isothiocyanate
(1.04 mmol) in dry THF (5 mL) over 3 min. After the addition,
the reaction mixture was allowed to warm to room tempera-
ture and was stirred for 24 h. The solvent was evaporated,
and the resulting residue was purified to give an analytically
pure sample of the corresponding heteroarotinoid.
Eth yl 4-{[N-(2,2,4,4-Tetr a m eth ylch r om a n -6-yl)ca r ba m -
oyl]a m in o}ben zoa te (13). The ester was prepared from
amine 18a and 4-ethoxycarbonylphenyl isocyanate (199 mg,
1.04 mmol). Evaporation of the solvent gave a white solid,
which was recrystallized (EtOAc) to afford 13 (261 mg, 66%)
as a white solid: mp 234-235 °C; IR (KBr) 3346, 3195, 1713,
1655 cm-1 1H NMR (DMSO-d6) δ 1.28 (m, 15 H), 1.80 (s, 2
;
H), 4.29 (q, J ) 7.09 Hz, 2 H), 6.65 (d, J ) 7.99 Hz, 1 H), 7.09
(dd, J ) 7.97, 1.98 Hz, 1 H), 7.44 (d, J ) 2.01 Hz, 1 H), 7.58
(d, J ) 8.08 Hz, 2 H), 7.88 (d, J ) 8.06 Hz, 2 H), 8.52 (s, 1 H),
8.99 (s, 1 H); 13C NMR (DMSO-d6) ppm 14.23, 28.10, 30.61,
32.50, 48.35, 60.20, 73.84, 117.10, 117.41, 117.51, 118.57,
122.41, 130.33, 131.29, 132.10, 144.56, 147.48, 152.33, 165.43.
Anal. (C23H28N2O4) C, H, N.
Eth yl 4-{[N-(2,2,4,4-Tetr a m eth ylch r om a n -6-yl)th ioca r -
ba m oyl]a m in o}ben zoa te (9). The compound was prepared
from amine 29a and 4-ethoxycarbonylphenyl isothiocyanate
(215 mg, 1.04 mmol). Evaporation of the solvent gave a tan,

Heteroarotinoids as Antagonists of M. bovis BCG
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1013
[(4-Nitr op h en yl)a m in o][(1,2,2,4-tetr a m eth yl(1,2-d ih y-
d r oqu in ol-6-yl))a m in o]m eth a n e-1-th ion e (14). To a chilled
(-5 °C) solution of (1,2,2,4-tetramethyl-1,2-dihydroquinol-6-
yl)amine (32, 150 mg, 0.74 mmol) dissolved in dry THF (5 mL)
was added dropwise 4-nitrophenyl isothiocyanate (141 mg, 7.78
mmol, 1.05 equiv) dissolved in dry THF (5 mL, 30 min). After
the addition, the reaction mixture was allowed to warm to
room temperature and was then stirred for 24 h. The solvent
was evaporated, and the resulting solid was recrystallized
(EtOAc:hexane, 1:1) to afford 14 (184 mg, 65%) as an orange-
133.08, 141.86, 144.70, 165.96, 179.21. Anal. (C23H28N2O2S2)
C, H, N, S.
[(6-Meth oxy-1,1,4,4-tetr am eth ylisoch r om an -5-yl)am in o]-
[(4-n itr op h en yl)a m in o]m eth a n e-1-th ion e (18). To a cooled
(-5 °C) solution of (6-methoxy-1,1,4,4-tetramethylisochroman-
5-yl)amine [39, 200 mg, 0.85 mmol] dissolved in dry THF (5
mL) in oven-dried equipment was added dropwise (1 h)
4-nitrophenyl isothiocyanate (160.7 mg, 8.92 mmol, 1.05
equiv). After the addition, the reaction mixture was allowed
to warm to room temperature and was then stirred (24 h). The
solvent was evaporated, and resulting solid was recrystallized
(pentane:H2CCl2, 1:1) to afford 18 (251 mg, 71%) as a light
yellow solid: mp 172-173.5; IR (KBr pellet) 3338, 3181 cm-1
;
1H NMR (DCCl3) δ 1.39 (s, 6 H), 1.93 (s, 3 H), 2,82 (s, 3 H),
5.30 (s, 1 H), 6.52 (d, 1 H, J ) 8.7 Hz), 6.92 (d, 1 H, J ) 2.4
Hz), 7.02 (q, 1 H, J ) 2.4 Hz, J ) 8.7 Hz), 7.67 (bs, 1 H), 7.77
(d, 2 H, J ) 9.0 Hz), 7.87 (bs, 1 H), 8.19 (d, 2 H, J ) 9.0 Hz);
13C NMR (DCCl3) ppm 18.52, 27.89, 30.91, 56.85, 111.08,
121.61, 122.81-145.44, 179.65. TLC analysis for C20H22N4O2S
showed one spot in the following solvent systems: hexane:
diethyl ether:H2CCl2, (1:1:1), Rf 0.40; chloroform:pentane, (2:
1), Rf 0.19; hexane:EtOAc, (2:1), Rf 0.14. Anal. (C20H22N4O2S‚
1.37 H2O) C, H, N.
yellow solid: mp 181-182 °C; IR (KBr pellet) 3368, 3214 cm-1
;
1H NMR [D3C(O)CD3] δ 1.24 (s, 6 H), 1.46 (s, 6 H), 3.56 (s, 2
H), 3.87 (s, 3 H), 7.01 (d, 1 H, J ) 8.7 Hz), 7.25 (d, 1 H, J )
8.7 Hz), 7.60 (bs, 1 H), 8.01 (d, 2 H, J ) 8.5 Hz), 8.15 (d, 2 H,
J ) 8.5 Hz), 8.48 (bs, 1 H); 13C NMR [D3C(CO)CD3] ppm 18.06,
26.72, 28.91, 34.54, 56.10, 75.55, 108.65, 122.11-145.87,
181.15. Anal. (C21H25O4N3S) C, H, N, S.
Eth yl 4-{[N-(2,2,4,4-Tetr a m eth ylch r om a n -8-yl)th ioca r -
ba m oyl]a m in o}ben zoa te (19). The ester was prepared from
amine 29b and 4-ethoxycarbonylphenyl isothiocyanate (215
mg, 1.04 mmol). Evaporation of the solvent gave a tan, viscous
oil, which was subjected to flash chromatography (Et2O:
hexanes, 1:1) to afford 19 (412 mg, 88%) as a white solid: mp
Eth yl 4-{[N-(2,2,4,4-Tetr a m eth ylth ioch r om a n -6-yl)ca r -
ba m oyl]a m in o}ben zoa te (15). A solution of 26 (400 mg, 1.81
mmol) in dry THF (8 mL) was treated dropwise with 4-ethoxy-
carbonylphenyl isocyanate (380 mg, 1.99 mmol) in dry THF
(8 mL) at 0-5 °C. The reaction was warmed to room temper-
ature and was stirred overnight. Evaporation of the solvent
gave a solid which was recrystallized (pentane:H2CCl2, 1:2) to
give 15 (480 mg, 63%) as a white solid: mp 200-201 °C; IR
1
56-58 °C; IR (KBr) 3325, 3199, 1715 cm-1; H NMR (DCCl3)
δ 1.29 (m, 15 H), 1.83 (s, 2 H), 4.38 (q, J ) 7.12 Hz, 2 H), 6.94
(m, 1 H), 7.15 (dd, J ) 7.91, 1.98 Hz, 1 H), 7.51 (d, J ) 7.99
Hz, 2 H), 7.98 (m, 1 H), 8.07 (d, J ) 8.02 Hz, 2 H), 8.30 (bs, 1
H), 8.40 (bs, 1 H); 13C NMR (DCCl3) ppm 14.26, 28.47, 31.02,
32.57, 48.71, 61.02, 75.98, 120.14, 120.49, 123.01, 124.06,
126.38, 127.64, 130.93, 132.27, 141.45, 143.91, 165.73, 177.83.
Anal. (C23H28N2O3S) C, H, N, S.
1
(KBr) 1688 cm-1; H NMR (DCCl3) δ 1.33 (s, 6 H), 1.37 (t, 3
H,), 1.38 (s, 6 H), 1.89 (s, 2 H), 4.35 (q, 2 H), 6.92 (dd, 1 H),
7.06 (s, 1 H), 7.26 (d, 1 H), 7.36 (d, 2 H), 7.43 (d, 2 H), 7.52 (s,
1 H), 7.92 (s, 2 H); 13C NMR (DCCl3) ppm 14.20, 31.46, 32.25,
35.66, 42.07, 54.10, 60.91, 118.49, 120.01, 120.66, 124.80,
128.93, 129.02, 130.97, 134.49, 142.95, 144.23, 153.35, 166.73.
Anal. (C23H28N2O3S) C, H, N, S.
N-{[(6-Met h oxy-1,1,4,4-t et r a m et h ylisoch r om a n -5-yl)-
a m in o]th ioxom eth yl}(4-n itr op h en yl)ca r boxa m id e (20).
To a cooled (-5 °C) solution of (6-methoxy-1,1,4,4-tetramethyl-
isochroman-5-yl)amine [39, 200 mg, 0.85 mmol] dissolved in
dry THF (5 mL) in oven-dried equipment was added dropwise
(1 h) ethyl (4-nitrophenyl)oxomethane isocyanate (186 mg, 8.92
mmol, 1.05 equiv) dissolved in dry THF (5 mL). After the
addition, the reaction mixture was allowed to warm to room
temperature and was then stirred (24 h). The solvent was
evaporated, and the resulting solid was recrystallized (pentane:
HCCl3, 1:2) to afford 20 (305 mg, 81%) as a yellow solid: mp
179 °C (dec); IR (KBr pellet) 3229, 3171, 1686 cm-1; 1H NMR
[D3C(O)CD3] δ 1.33 (s, 6 H), 1.56 (s, 6 H), 3.57 (s, 2 H), 3.80 (s,
3 H), 6.85 (d, 1 H, J ) 8.3 Hz), 7.10 (d, 1 H, J ) 8.3 Hz), 8.10
(d, 1 H, J ) 7.8 Hz), 8.39 (dd, 1 H, J ) 7.8 Hz), 10.85 (s, 1 H),
11.82 (s, 1 H); 13C NMR [D3C(O)CD3] ppm 24.10, 26.67, 34.55,
56.05, 71.34, 111.19, 124.24-135.77, 168.77, 182.56. Anal.
(C22H25N3O5S‚0.6H2O) C, H, N, S.
Eth yl 4-{[(1,2,2,4-Tetr a m eth yl(1,2-d ih yd r oqu in ol-6-yl)-
ca r ba m oyl]a m in o]ben zoa te (16). A solution of (1,2,2,4-
tetramethyl-1,2-dihydroquinol-6-yl)amine (27, 150 mg, 0.74
mmol) dissolved in dry THF (4 mL) in oven-dried equipment
was cooled to -5 °C (ice and NaCl), and ethyl 4-isocyanato-
benzoate (148.5 mg, 7.78 mmol, 1.05 equiv) dissolved in dry
THF (5 mL) was then added dropwise (30 min). After the
addition, the reaction mixture was allowed to warm to room
temperature and was then stirred for 24 h. The solvent was
evaporated, and the solid was recrystallized (pentane:HCCl3,
1:1) to afford 16 (206 mg, 71%) as a white, flaky solid: mp
1
211-212 °C; IR (KBr pellet) 3352, 3262, 1709 cm-1; H NMR
(DCCl3) δ 1.29 (s, 6 H), 1.36 (t, 3 H), 1.63 (bs, 1 H), 1.94 (s, 3
H), 2,87 (s, 3 H), 4.37 (q, 2 H), 5.33 (s, 1 H), 6.47 (d, 1 H, J )
8.3 Hz), 6.67 (bs, 1 H), 6.97 (d, 1 H, J ) 1.9 Hz), 7.13 (q, 1 H,
J ) 1.9 Hz, J ) 8.3 Hz), 7.4 (d, 2 H, J ) 9.0 Hz), 7.92 (d, 2 H,
J ) 9.0 Hz); 13C NMR (DCCl3) ppm 14.33, 18.50, 27.21, 30.75,
56.39, 60.70, 111.11, 118.14, 120.64-143.96, 154.23, 180.45.
TLC analysis for C23H27N3O3 showed one spot in the following
solvent systems: hexane:diethyl ether:H2CCl2, (1:1:1), Rf 0.46;
chloroform:pentane, (2:1), Rf 0.40; hexane:EtOAc, (2:1), Rf 0.18.
Anal. (C23H27N3O3‚0.3H2O) C, H, N.
[(4-Nitr op h en yl)a m in o][(2,2,4,4-tetr a m eth ylch r om a n -
8-yl)a m in o]m eth a n e-1-th ion e (21). Ether 21 was prepared
from amine 29b and 4-nitrophenyl isothiocyanate (187 mg,
1.04 mmol). Evaporation of the solvent gave a yellow oil, which
was subjected to flash chromatography (Et2O:hexanes, (2:1)
to afford 21 (316 mg, 82%) as a yellow solid: mp 160-161 °C;
1
IR (KBr) 3327, 3232 cm-1; H NMR (DCCl3) δ 1.35 (s, 6 H),
1.36 (s, 6 H), 1.87 (s, 2 H), 6.96 (m, 1 H), 7.22 (dd, J ) 8.02,
2.04 Hz, 1 H), 7.62 (m, 1 H), 7.68 (d, J ) 8.07 Hz, 2 H), 8.21
(d, J ) 8.07 Hz, 2 H), 8.30 (bs, 1 H), 8.34 (bs, 1 H); 13C NMR
(DCCl3) ppm 28.51, 31.09, 32.61, 48.67, 76.26, 120.48, 12.37,
122.53, 124.79, 125.24, 125.47, 133.10, 143.91, 144.21, 144.80,
178.21. Anal. (C20H23N3O3S) C, H, N, S.
N-4-[2,3-Dih yd r o-2,2,4,4-t et r a m et h yl-2H-1-b en zot h io-
p yr a n -6-yl)-N-(4-eth oxyca r bon ylp h en yl)u r ea (17). A solu-
tion of crude amine 26 (400 mg, 1.70 mmol) in THF (10 mL)
was treated dropwise with 4-ethoxycarbonylphenyl isocyanate
(412 mg, 1.99 mmol) in THF (10 mL) at 0-5 °C. The reaction
mixture was allowed to warm to room temperature and was
stirred overnight. Evaporation of the solvent gave a solid.
Recrystallization (pentane:CH2Cl2, 6:1) gave 17 (710 mg, 92%)
as a white solid: mp 134-135 °C; IR (KBr) 1688 cm-1; 1H NMR
(DCCl3) δ 1.38 (s, 6 H), 1.40 (t, 3 H, J ) 7.2 Hz), 1.43 (s, 6 H),
1.95 (s, 2 H), 4.35 (q, 2 H, J ) 7 Hz), 7.04 [dd, 1 H, Ar-H],
7.16 [s, 1 H, Ar-H], 7.36 [d, 1 H, Ar-H], 7.55 [d, 2 H, Ar-H],
8.00 [s, 1 H, NH], 8.02 [d, 2 H, Ar-H], 8.35 [s, 1 H, NH]; 13C
NMR (DCCl3) ppm 14.16, 31.46, 32.35, 35.67, 42.25, 53.72,
60.97, 122.95, 123.17, 124.05, 127.72, 129.27, 130.65, 132.97,
Eth yl 4-{[N-(2,2,4,4-Tetr a m eth ylch r om a n -8-yl)ca r ba m -
oyl]a m in o}ben zoa te (22). Ether 22 was prepared from
amine 29b (0.130 g, 0.63 mmol) and 4-ethoxycarbonylphenyl
isocyanate (199 mg, 1.04 mmol). Evaporation of the solvent
gave a tan residue, which was flash chromatographed [Et2O:
hexanes (8:1)] to afford 22 (332 mg, 84%) as a white solid: mp
1
174-176 °C; IR (KBr) 3348, 3201, 1715, 1675 cm-1; H NMR
(DCCl3) δ 1.27 (m, 15 H), 1.78 (s, 2 H), 4.34 (q, J ) 7.21 Hz, 2
H), 6.87 (m, 1 H), 6.97 (dd, J ) 8.03, 1.98 Hz, 1 H), 7.44 (d, J
) 8.07 Hz, 2 H), 7.74 (s, 1 H), 7.91 (d, J ) 7.98 Hz, 2 H), 7.95

1014 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Brown et al.
(d, J ) 1.99 Hz, 1 H), 8.03 (s, 1 H); 13C NMR (DCCl3) ppm
14.28, 28.41, 30.97, 32.53, 49.02, 60.28, 75.45, 117.31, 118.26,
120.49, 120.89, 124.21, 127.61, 130.80, 131.36, 141.45, 143.39,
152.80, 166.66. Anal. (C23H28N2O4) C, H, N.
103.67, 107.42, 116.62, 124.29, 124.87, 127.41, 154.22, 156.34.
Anal. (C12H14O2) C, H.
1,2,2,4-Tetr a m eth yl-1,2-d ih yd r oqu in olin e (27). A mix-
ture of powdered KOH (299 mg, 5.7 mmol) dissolved in DMSO
(10 mL) was stirred at room temperature until all KOH
dissolved, and then the temperature was adjusted to 10 °C.
2,2,4-Trimethyl-1,2-dihydroquinoline (30, 1.00 g, 5.77 mmol)
dissolved in DMSO (5 mL) was added dropwise, followed
immediately by the addition of CH3I (1.09 g, 7.7 mmol). The
reaction mixture was stirred (0.5 h) and then was poured into
ice-cold water (10 mL). Extracts with H2CCl2 (3 × 5 mL) were
combined and washed with water (1 × 10 mL) and brine (1 ×
10 mL) and then dried (Na2SO4). Evaporation of the solvent
and flash chromatography (silica gel, hexanes) of the residue
2,2,4,4-Tet r a m et h ylch r om a n (23). To 3.0 M methyl-
magnesium bromide in diethyl ether (121 mL, 0.36 mol), along
with dry THF (240 mL), was added dropwise (room temper-
ature) 4,4-dimethylcoumarin 1420 (16.00 g, 90.8 mmol) dis-
solved in THF (10 mL), and the resulting mixture was stirred
at reflux for 4 days. The mixture was allowed to cool to room
temperature and then was cooled to 0 °C (ice bath). Saturated,
aqueous NH4Cl solution (820 mL) was added, the resulting
aqueous layer was extracted (Et2O, 4 × 150 mL), and the
combined organic extracts were washed with H2O (150 mL)
and brine (150 mL) and then were dried (Na2SO4). Evaporation
of the solvent gave a pale yellow oil, which crystallized on
standing. Recrystallization (petroleum ether) of the yellow
solid gave 4-(2-hydroxyphenyl)-2,4-dimethyl-2-pentanol (16.00
g, 84%) as clear, needlelike crystals: mp 88-90 °C (lit.16 mp
afforded 27 as a yellow oil (0.784 g, 71%): IR (neat) 1048 cm-1
;
1H NMR (DCCl3) δ 1.37 (s, 6 H), 2.23 (s, 3 H), 2,96 (s, 3 H),
5.36 (s, 1 H), 6.87-7.24 (m, 4 H); 13C NMR (DCCl3) ppm 18.64,
25.74, 30.60, 54.53, 111.43, 112.5, 118.12-144.56. The com-
pound was used immediately to prepare 32 and/or 33.
1
91 °C); IR (KBr) 3406, 1731 cm-1; H NMR (DCCl3) δ 1.17 (s,
2,2,4,4-Tetr a m eth yl-6-n itr och r om a n (28a ) a n d 2,2,4,4-
Tetr a m eth yl-8-n itr och r om a n (28b). To a solution of the
chroman 2317 (5.00 g, 26.3 mmol) in freshly distilled Ac2O (5
mL) at -5 °C (ice/salt) was added dropwise a mixture of cold,
concentrated HNO3 (1.89 mL) and Ac2O (4.42 mL) over 10 min.
After being stirred at -5 °C (90 min), the reaction mixture
was then poured into saturated, aqueous NaHCO3 (50 mL).
The resulting emulsion was extracted (H2CCl2, 3 × 30 mL),
and the combined extracts were washed with H2O (40 mL) and
brine (40 mL) and were then dried (Na2SO4). Evaporation of
the solvent gave a dark brown liquid, which was subjected to
flash chromatography (hexanes:Et2O, 20:1) to provide isomers
28a and 28b (isomer 28a was collected in fractions 5-12, and
isomer 28b was collected in fractions 15-20). The 6-isomer
28a (2.67 g, 43%) was isolated as a pale yellow solid: mp 56-
6 H), 1.41 (s, 6 H), 2.24 (s, 2 H), 6.58 (dd, J ) 7.21, 1.56 Hz, 1
H), 6.87 (m, 1 H), 7.04 (m, 1H), 7.35 (dd, J ) 7.18, 1.59 Hz, 1
H); 13C NMR (DCCl3) ppm 30.89, 30.94, 37.50, 52.31, 73.43,
117.15, 120.89, 127.50, 127.71, 133.98, 155.05. Previous data
on this diol were an elemental analysis16 and some spectral
information on a crude sample.20
A mixture of the above diol18 (45.02 g, 0.22 mol), 85% H3PO4
(21.21 mL, 0.18 mol), and benzene (225 mL) was stirred and
heated (80 °C). After ∼5 min, the first of 3 portions of P2O5
(25.42 g, 0.54 mol) was added. The other 2 portions of P2O5
were added at ∼7 h intervals for a total of 76.26 g (3 × 25.42
g), and the reaction mixture was allowed to reflux (24 h). The
reaction mixture was allowed to cool to room temperature, and
the benzene solution was decanted from a dark, purple residue
in the bottom of the reaction flask. The residue was simply
rinsed (ether, 3× 150 mL), and the ether extracts were
combined with the benzene solution. The combined organic
extracts were washed with 5% aqueous NaHCO3 (3 × 100 mL)
and brine (3 × 100 mL) and were then dried (Na2SO4). The
solvent was evaporated to a liquid which was subsequently
vacuum distilled to afford 2,2,4,4-tetramethylchroman (23)
(21.5 g, 54%) as a colorless oil: bp 69-71 °C/1.5 mmHg (lit.16
1
58 °C; IR (KBr) 1581, 1341 cm-1; H NMR (DCCl3) δ 1.39 (s,
6 H), 1.41 (s, 6 H), 1.89 (s, 2 H), 6.85 (d, J ) 7.93 Hz, 1 H),
7.98 (dd, J ) 7.01, 1.98 Hz, 1 H), 8.22 (d, J ) 1.97 Hz, 1 H);
13C NMR ppm 28.48, 31.12, 32.69, 48.07, 76.39, 118.45, 123.18,
123.54, 131.93, 141.38, 158.46. The 8-isomer 28b (1.61 g, 26%)
was obtained as a creamy white solid: mp 52-54 °C; IR (KBr)
1
1582, 1341 cm-1; H NMR (DCCl3) δ 1.37 (s, 6 H), 1.39 (s, 6
H), 1.91 (s, 2 H), 6.94 (m, 1 H), 7.48 (dd, J ) 7.63, 1.87 Hz, 1
H), 7.57 (dd, J ) 7.59, 1.83 Hz, 1 H); 13C NMR (DCCl3) ppm
28.35, 31.30, 32.55, 48.33, 76.86, 118.45, 119.52, 122.68,
130.86, 135.12, 146.89, 146.89. Both isomers were immediately
used to prepare 29a and 29b, respectively.
1
bp 102-104 °C); H NMR (DCCl3) δ 1.34 (s, 6 H), 1.35 (s, 6
H), 1.84 (s, 2 H), 6.79 (dd, J ) 7.68, 1.79 Hz, 1 H), 6.85 (m, 1
H), 7.08 (m, 1 H), 7.31 (dd, J ) 7.76, 1.83 Hz, 1 H); 13C NMR
(DCCl3) ppm 28.45, 30.74, 32.70, 49.20, 74.30, 117.97, 120.66,
126.82, 127.05, 131.63, 152.62. The only prior information on
chroman 23 was an elemental analysis.16
2,2,4,4-Tetr a m eth yl-6-a m in och r om a n (29a ) a n d 2,2,4,4-
Tetr a m eth yl-8-a m in och r om a n (29b). A mixture of nitro
compound 28a (1.60 g, 6.8 mmol) or 28b, iron powder (1.36 g,
24.3 mmol, Sigma-Aldrich Chemical Co.), glacial acetic acid
(2.86 g, 47.6 mmol), and absolute EtOH (17 mL) was stirred
at reflux (12 h). The reaction was allowed to cool to room
temperature and poured into H2O (68 mL), and the resulting
emulsion was extracted with Et2O (2 × 65 mL) and HCCl3 (3
× 65 mL). The combined organic extracts were washed with
H2O (3 × 65 mL), dried (Na2SO4), and concentrated to a dark
oil. This crude mixture was then dissolved in Et2O (45 mL),
and the resulting solution was extracted (2 N HCl, 2 × 55 mL).
The acid solution was neutralized with 40% Na2CO3 (pH ∼
8), and the resulting solution was extracted (Et2O, 2 × 55 mL).
The combined organic extracts were dried (Na2SO4), and the
solvent was evaporated to amine 29a (or 29b) as an oil which
crystallized upon standing at room temperature. The 6-isomer
29a (586 mg, 42%) was obtained as an off-white solid: mp 40-
2,2,4-Tr im eth yl-2H-1-ben zop yr a n -7-ol (25). To 3.0 M
methylmagnesium bromide in diethyl ether (30 mL, 90.8
mmol), along with dry THF (60 mL), was added dropwise (room
temperature) coumarin 3717 (4.00 g, 22.7 mmol) dissolved in
THF (25 mL), and the resulting mixture was stirred at room
temperature (2 days). During the addition, some heat evolved,
and a yellow precipitate formed. The reaction was quenched
with saturated, aqueous NH4Cl (250 mL), and the resulting
mixture was separated into two layers. The aqueous layer was
extracted (Et2O, 5 × 50 mL), and the combined organic extracts
were dried (Na2SO4). Evaporation of the solvent afforded a
dark orange oil which was dissolved in 20 mL of glacial acetic
acid, and the resulting solution was stirred and heated gently
(slightly below the boiling point of acetic acid) for 2 h. The
reaction mixture was allowed to cool to room temperature and
was poured into H2O (100 mL). The resulting emulsion was
extracted (Et2O, 5 × 50 mL), and the combined organic extracts
were washed with saturated, aqueous NaHCO3 (3 × 50 mL)
and brine (2 × 50 mL) and were dried (Na2SO4). Evaporation
of the solvent gave a dark brown, viscous oil, which was
subjected to flash chromatography (ether:hexanes, 5:1) that
provided 25 (1.51 g, 40%) as a white solid: mp 128-130 °C;
IR (KBr) 3330 cm-1; 1H NMR (DCCl3) δ 1.38 (s, 6 H), 1.96 (d,
J ) 2.09 Hz, 3 H), 5.17 (bs, 1 H), 5.28 (m, 1 H), 6.32 (d, J )
2.01 Hz, 1 H), 6.35 (dd, J ) 8.01, 2.0 Hz, 1 H), 6.99 (d, J )
8.03 Hz, 1 H); 13C NMR (DCCl3) ppm 17.92, 28.03, 76.38,
1
42 °C; IR (KBr) 3433, 3357 cm-1; H NMR (DCCl3) δ 1.30 (s,
6 H), 1.31 (s, 6 H), 1.78 (s, 2 H), 3.29 (bs, 2 H), 6.46 (dd, J )
8.03, 1.99 Hz, 1 H), 6.61 (d, J ) 1.80 Hz, 1 H), 6.63 (d, J )
7.89 Hz, 1 H); 13C NMR (DCCl3) ppm 28.35, 30.96, 32.57, 49.32,
73.81, 113.41, 114.88, 118.29, 132.28, 139.51, 145.35.
It was discovered that a crude mixture of the isomeric nitro
compounds 29a and 29b could be coreduced by the above
method. The final mixture of amines 29a and 29b was more
easily separated by flash column chromatography (hexanes:
Et2O, 1:1) than the corresponding mixture of nitro compounds

Heteroarotinoids as Antagonists of M. bovis BCG
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 1015
(fractions 4-10 yielded 29b and fractions 17-25 yielded 29a ).
The 8-isomer 29b (430 mg, 31%) was obtained as a very light
pink solid: mp 43-45 °C; IR (KBr) 3455, 3360 cm-1; 1H NMR
(DCCl3) δ 1.32 (s, 6 H), 1.36 (s, 6 H), 1.83 (s, 2 H), 3.54 (bs, 2
H), 6.54 (dd, J ) 7.94, 2.02 Hz, 1 H), 6.70 (m, 2 H); 13C NMR
(DCCl3) ppm 28.63, 30.96, 32.58, 49.34, 74.48, 112.37, 116.03,
120.24, 131.33, 136.11, 140.07. Amines 29a and 29b were used
at once to prepare 9, 11, and 13 as well as 19, 21, and 22,
respectively.
[1,2,2,4-Tetr am eth yl-1,2-dih ydr oqu in ol-6-yl]am in e (32).
To 1,2,2,4-tetramethyl-1,2-dihydroquinoline (27, 1.00 g, 5.3
mmol) dissolved in a 3 M solution of LiClO4 in diethyl ether
(5 mL) was added dropwise freshly prepared bis(2,2,2-tri-
chloroethyl) azodicarboxylate (31, 4.5 g, 11.8 mmol) dissolved
in diethyl ether (5 mL) at 0 °C. The solution was then carefully
warmed to 55 °C and stirred at this temperature (3 h). The
new reaction mixture was cooled to 0 °C, and ice water (5 mL)
was added. Extraction with H2CCl2 (3 × 10 mL), followed by
washing the extracts with brine (1 × 5 mL) and drying
(Na2SO4), yielded an aryl azide (2.58 g, 85%). This aryl azide
was reduced, without purification, by dissolving it in concen-
trated acetic acid (5 mL). To this solution was added ap-
proximately 1 equiv by weight of Zn dust (2.6 g). The reaction
mixture was stirred for 15 min, and 9 µL of acetone was added.
After stirring the reaction mixture (3 h) at room temperature,
the new mixture was filtered through Celite. Then a saturated,
aqueous solution of NaHCO3 (5 mL) was added, and the
mixture was extracted (H2CCl2, 3 × 10 mL). Flash chroma-
tography of the concentrated extracts with silica gel (hexane:
EtOAc, 1:1) gave solid 32 (546 mg, 51% from 27): mp 87-89
2,2,4-Tr im eth yl-1,2-d ih yd r oqu in olin e (30). A mixture of
freshly distilled aniline (20.0 g, 0.21 mol) together with a
catalytic amount of iodine (0.3 g) and concentrated HCl (0.2
mL) were heated to 155 °C, and then acetone (∼ 400 mL) was
added slowly at such a rate so that the temperature of the
mixture did not fall below 140 °C. Unreacted acetone and H2O
(a reaction byproduct) distilled off during the addition process.
After another addition of acetone (250 mL, ∼3.5 h), the
reaction mixture was stirred (1 h) and was then allowed to
cool to room temperature. Extraction with hexane (3 × 100
mL) was followed by washing the combined organic extracts
with H2O (1 × 100 mL) and brine (1 × 100 mL) and then
drying (MgSO4). The hexane was evaporated, and the resulting
product was purified by distillation (bp 109-111 °C/0.75
mmHg) to yield 30 as a pale yellow oil (27.9 g, 76%): IR (neat)
1
°C; IR (neat) 3338, 3224 cm-1; H NMR (DCCl3) δ 1.30 (s, 6
H), 1.97 (s, 3 H), 2,78 (s, 3 H), 5.38 (s, 1 H), 6.42 (d, 1 H, J )
9.3 H), 6.48 (d, 1 H, J ) 2.3 Hz), 6.55 (q, 1 H, J ) 2.1 Hz, J )
9.3 Hz), 6.56 (s, 1 H), 6.8 (bs, 1 H); 13C NMR (DCCl3) ppm
18.53, 25.70, 30.68, 55.60, 111.91, 112.14, 115.72-138.78.
Amine 32 was used at once to prepare 12, 14, and 16.
1
3301 [N-H] cm-1; H NMR (DCCl3) δ 1.41 (s, 6 H), 2.18 (s, 3
H), 3.79 (bs, 1 H), 5.45 (s, 1 H), 6.55-7.15 (m, 4 H); 13C NMR
(DCCl3) ppm 18.64, 23.44, 54.34, 110.53, 111.91, 116.12-
145.07. The compound 30 was used immediately to prepare
27.
1,2,2,4-Tetr a m eth yl-1,2-d ih yd r oqu in olin e-6-ca r ba ld e-
h yd e (33). Phosphorus oxychloride (4.1 g, 0.026 mol) was
added dropwise to DMF (12 mL) at 0 °C. After the reaction of
OPCl3 with DMF had subsided, 1,2,2,4-tetramethyl-1,2-dihy-
droquinoline [(31), 5 g, 0.026 mol] dissolved in DMF (30 mL)
was slowly added at 0 °C. The reaction mixture was allowed
to stir (24 h) at room temperature and was then cooled to 0
°C, after which cold water (5 mL) was carefully added. The
reaction mixture was extracted with methylene chloride (3 ×
30 mL), and the combined organic extracts were washed with
water (1 × 10 mL) and brine (1 × 10 mL) and dried (MgSO4).
After evaporation of the solvents and refrigeration of the oil
(24 h), aldehyde 33 crystallized as a pale yellow solid (no
purification required, 3.57 g, 64%): mp 39-41 °C; IR (pellet)
Bis(2,2,2-tr ich lor oeth yl) Azod ica r boxyla te (31). It was
discovered that fresh and highly purified 31 was required in
the preparation of 32. Slightly crude commercial material and
the long preparation in Organic Syntheses21 for 31 prompted
us to modify the latter approach. A solution of 1.0 g (0.023
mol) of 85% hydrazine hydrate in 6 mL of 95% ethanol was
cooled in an ice bath, and 9.6 g (0.046 mol) of 2,2,2-trichloro-
ethyl chloroformate was added dropwise so that the temper-
ature was kept below 20 °C. During the addition of 1 equiv of
the chloroformate, a white precipitate formed. After one-half
of the chloroformate had been added, a solution of sodium
carbonate (2.5 g, 0.024 mol) in water (10.0 mL) was added
dropwise (2 h) along with the remaining chloroformate. The
rate of addition of these two reagents was such that the flow
of the chloroformate was faster than that of the sodium
carbonate so that an excess of chloroformate was present at
all times. The temperature was kept below 20 °C during the
addition. As the second equivalent of chloroformate was added,
the white precipitate dissolved. After the addition of the
reactants was complete (4 h), the reaction was allowed to stir
for an additional 30 min while the solution warmed to room
temperature. The reaction mixture was then transferred to a
separatory funnel, and the viscous, organic layer (bottom) was
separated from the aqueous layer. The organic layer was
dissolved in ether (20 mL), and the reaction vessel was washed
with ether (10 mL). This ether washing was used to extract
the aqueous layer again. The ether extracts were combined,
dried (MgSO4), and filtered, which was followed by evaporation
of the solvent.
Bromine (1.6 g, 20.0 mmol) in dichloromethane (150 mL)
was added dropwise (1 h) to a solution of dichloromethane (500
mL), hydrazide (7.0 g, 18.2 mmol, 85%), and pyridine (1.50 g,
20.0 mmol), and solution was cooled to 0 °C (ice bath) under
argon. The reaction mixture turned from colorless to yellow
upon the addition. The reaction was complete after 30-60 min
at room temperature, as determined by TLC (silica gel, EtOAc:
hexane, 1:1). The reaction mixture was then diluted to 1000
mL with dichloromethane and washed with 5% HCl (2 × 300
mL), saturated sodium bicarbonate (300 mL), water (3 × 300
mL), and saturated NaCl (1 × 300 mL) to give diazo compound
31 as light yellow solid [4.26 g, 56%]: mp 115-116 °C (lit.21
mp 109-110.5 °C); IR (KBr pellet) 1786, 1723 cm-1; 1H NMR
(DCCl3) δ 5.08 (s, 4 H); 13C NMR (DCCl3) ppm 77.45, 93.09,
158.42. Diazo compound 31 was used immediately to prepare
32.
1
2733, 1669 cm-1; H NMR (DCCl3) δ 1.38 (s, 6 H), 2.03 (s, 3
H), 2.91 (s, 3 H), 5.30 (s, 1 H), 6.50 [d, 1 H, J ) 8.4 Hz, Ar-H],
7.52 [d, 1 H, J ) 2.4 Hz, Ar-H], 7.52 [q, 1 H, J ) 8.4 Hz, J )
2.4 Hz, Ar-H], 9.67 (s, 1 H); 13C NMR (DCCl3) ppm 18.57, 28.75,
31.21, 57.60, 109.14, 121.76-150.04, 190.19. Aldehyde 33 was
used at once to prepare 34.
4-Meth yl-6-(1,2,2,4-tetr a m eth yl(1,2-d ih yd r oqu in olin -6-
yl))-5,6-d ih yd r op yr a n -2-on e (34). To a solution of ethyl 3,3-
dimethylacrylate (205 mg, 1.5 mmol) and dry THF (3 mL) was
added dropwise LDA (0.51 mL, 1.53 mmol, 3 M solution in
toluene) at -78 °C. Following the addition, the reaction
mixture was stirred (1 h), after which 33 (0.3 g, 1.46 mmol)
dissolved in dry THF (2 mL) was added. After stirring the
reaction mixture for 1 h at -78 °C, the reaction was quenched
with a saturated, aqueous solution of ammonium chloride (1.5
mL) The resulting mixture was allowed to warm to room
temperature and was then stirred for an additional 1 h.
Extraction of the mixture with EtOAc (3 × 3 mL) was followed
by washing the combined organic extracts with water (1 × 1
mL) and brine (1 × 1 mL). After drying (MgSO4), the solvent
was removed, and a thick, dark-red oil was recovered as 34
1
(171 mg, 43%): IR (neat) 1725 cm-1; H NMR (DCCl3) δ 1.28
(s, 6 H), 2.03 (s, 3 H), 2.05 (s, 3 H), 2.91 (s, 3 H), 2.38 (dd, 2
H), 2.75, (d, 2 H), 5.25 (s, 1 H), 5.91 (s, 1 H), 6.50 (d, 1 H), 7.45
(m, 1 H); 13C NMR (DCCl3) ppm 18.57, 28.75, 31.21, 56.60,
109.14, 121.76-150.04, 168.35. Amine 34 was used im-
mediately to prepare 35.
4-Meth yl-6-(1,2,2,4-tetr a m eth yl(1,2-d ih yd r oqu in olin -6-
yl))-5,6-d ih yd r op yr a n -2-ol (35). To a chilled solution (-78
°C) of DIBAL-H in hexane (0.37 mL. 0.59 mmol, 1.6 M) was
added 34 (107 mg, 0.36 mmol) in dry THF (2 mL). The mixture
was stirred (20 min) and then quenched with 0.75 mL of a
saturated, aqueous solution of Rochelle salt (saturated solution

1016 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Brown et al.
of sodium and potassium tartrate, 1:1). After allowing the
reaction mixture to warm to room temperature, the mixture
was extracted with EtOAc (2 × 2 mL), and the extracts were
washed with water (1 × 1 mL) and brine (1 × 1 mL). After
drying (Na2SO4), the solvent was evaporated to give a thick,
red oil (35) [one product via TLC (hexane:EtOAc, 2:1) (92 mg,
85%)]. IR (neat) for 35 showed 3452 [O-H] cm-1. Compound
35 was used at once and without further purification to
generate 36.
37 °C for 14 days, and the number of viable bacteria was scored
by counting colonies. The experments were run separately and
in duplicate. The MIC value was defined as the lowest
concentration of drug resulting in 99% reduction in the number
of colonies on each plate compared to those on a plate free of
the agent at the same dilution of the culture suspension. The
technique had previously been utilized to demonstrate the
prowess of similar compounds in terms of inhibition of mycolic
acid synthesis.13
(2Z,4E)-3-Met h yl-5-(1,2,2,4-t et r a m et h yl(1,2-d ih yd r o-
qu in olin -6-yl))p en ta -2,4-d ien a l (36). To lactol 35 (150 mg,
0.5 mmol) dissolved in ClCH2CH2Cl (2 mL) was slowly added
5% HCl (2 mL). The reaction mixture was warmed to 55 °C (3
h) and monitored by TLC (hexane:EtOAc, 4:1) until completion.
The mixture was then cooled to room temperature and
carefully neutralized with a saturated, aqueous solution of
NaHCO3. The aqueous layer was extracted (H2CCl2; 2 × 3 mL),
and the combined extracts were washed with water (1 × 2 mL)
and brine (1 × 2 mL). After drying (MgSO4) and evaporation
of the solvent, the residue was purified over silica gel (hexane:
EtOAc, 1:1) to yield a bright red oil as 36 (87 mg, 62%): IR
(neat) 2785, 1655 cm-1; 1H NMR (DCCl3) δ 1.28 (s, 6 H), 2.03
(s, 3 H), 2.35 (s, 3 H, J ) 0.3 Hz), 2.91 (s, 3 H), 5.25 (s, 1 H),
5.91 (d, 1 H), 6.25 (d, 1 H), 6.50 (d, 1 H), 7.45 (m, 2 H), 10.15
(d, 1 H); 13C NMR (DCCl3) ppm 13.06, 18.57, 27.90, 30.21,
56.86, 110.14, 122.76-136.04, 191.04. Aldehyde 36 was used
at once to prepare 10.
Ack n ow led gm en t. We (K.D.B./D.M.B.) gratefully
acknowledge the partial support of this work by the
National Institutes of Health, National Cancer Institute
[CA-73639]. The antibacterial activities were deter-
mined via a program project (to P.J .B.) from the NIAID,
NIH, AI-46393. I (K.D.B.) am pleased to acknowledge
funding for the Varian Inova 400 MHz NMR spectrom-
eter in the Oklahoma Statewide Shared NMR facility
by the National Science Foundation [BIR-9512269], the
Oklahoma State Regents for Higher Education, the W.
M. Keck Foundation, and Conoco, Inc. Thanks are
extended to the College of A&S for salary support
(K.D.B.).
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6-Meth oxy-1,1,4,4-tetr am eth yl-5-n itr oisoch r om an e (38).
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1
solid: mp 82-83 °C; IR (KBr) 1241 cm-1; H NMR (DCCl3) δ
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