Analogs of N′-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl) methanimidamide inhibit Mycobacterium tuberculosis methionine aminopeptidases

Article abstract of DOI:10.1016/j.bmc.2012.05.022

Our previous target validation studies established that inhibition of methionine aminopeptidases (MtMetAP, type 1a and 1c) from Mycobacterium tuberculosis (Mtb) is an effective approach to suppress Mtb growth in culture. A novel class of MtMetAP1c inhibitors comprising of N′-hydroxy-N-(4H,5H- naphtho[1,2-d]thiazol-2-yl)methanimidamide (4c) was uncovered through a high-throughput screen (HTS). A systematic structure-activity relationship study (SAR) yielded variants of the hit, 4b, 4h, and 4k, bearing modified A- and B-rings as potent inhibitors of both MtMetAPs. Except methanimidamide 4h that showed a moderate Mtb inhibition, a desirable minimum inhibitory concentration (MIC) was not obtained with the current set of MtMetAP inhibitors. However, the SAR data generated thus far may prove valuable for further tuning of this class of inhibitors as effective anti-tuberculosis agents.

Full text of DOI:10.1016/j.bmc.2012.05.022

Bioorganic & Medicinal Chemistry 20 (2012) 4507–4513
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Analogs of N0-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide
inhibit Mycobacterium tuberculosis methionine aminopeptidases
Shridhar Bhat a, Omonike Olaleye a, ,à, Kirsten J. Meyer a,à, Wanliang Shi c, Ying Zhang c, Jun O. Liu a,b,
⇑
a Department of Pharmacology & Molecular Sciences, 725 North Wolfe Street, Baltimore, MD 21205, USA
b Department of Oncology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA
c W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 March 2012
Revised 3 May 2012
Accepted 11 May 2012
Available online 17 May 2012
Our previous target validation studies established that inhibition of methionine aminopeptidases (MtMe-
tAP, type 1a and 1c) from Mycobacterium tuberculosis (Mtb) is an effective approach to suppress Mtb
growth in culture. A novel class of MtMetAP1c inhibitors comprising of N0-hydroxy-N-(4H,5H-naph-
tho[1,2-d]thiazol-2-yl)methanimidamide (4c) was uncovered through a high-throughput screen (HTS).
A systematic structure–activity relationship study (SAR) yielded variants of the hit, 4b, 4h, and 4k, bear-
ing modified A- and B-rings as potent inhibitors of both MtMetAPs. Except methanimidamide 4h that
showed a moderate Mtb inhibition, a desirable minimum inhibitory concentration (MIC) was not
obtained with the current set of MtMetAP inhibitors. However, the SAR data generated thus far may
prove valuable for further tuning of this class of inhibitors as effective anti-tuberculosis agents.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Methionine aminopeptidase
Tuberculosis
N0-Hydroxy-N-(thiazol-2-
yl)methanimidamide
1. Introduction
in every life form. In prokaryotes, and in organelles like mitochon-
dria and plastids the start codon is translated by N-formylmethio-
It is estimated that as much as one-third of the world’s popula-
tion is infected with Mycobacterium tuberculosis (Mtb). Globally
mortality due to Mtb infection is second only to HIV/AIDS,1 and
an expanding epidemic of multi-drug resistant, extensively drug-
resistant, and totally drug-resistant2 tuberculosis (TB) coupled
with Mtb (including latent) and HIV co-infection is a serious health
threat requiring urgent attention.3 Since the approval of rifampin
as a treatment for TB by the US Food and Drug Administration
(FDA) in 1971, there has been a long hiatus with no new and sig-
nificant anti-TB drugs brought to the clinic. Recently there has
been an awakening to the urgency of the situation, and now there
are several candidate anti-TB drugs in various stages of develop-
ment.4 Continued emphasis on drug discovery is needed as new
drugs with novel modes of action are required to catch up with
the rapidly evolving resistant strains of Mtb. To this end we have
been actively pursuing inhibitors of Mtb methionine aminopepti-
dases (MetAPs) as novel targets.
nine, and a prerequisite step of deformylation by peptide
deformylase (PDF) is required.5 The importance of these two en-
zymes is underscored by the study by Solbiati et al. showing that
null mutations in PDF or MetAP gene are lethal to Escherichia coli.6
The genome of the H37Rv strain of Mtb contains two MetAP genes,
mapA and mapB encoding for Mtb methionine aminopeptidase 1a
(MtMetAP1a) and MtMetAP1c, respectively. We have previously
characterized these two isozymes both biochemically and by
X-ray crystallography.7 The crystal structure revealed the presence
of an SH3 domain at the N-terminal extension (40 amino acids) sug-
gesting a mode of association with the ribosome. MtMetAP1a iso-
zyme is a minimal protein exhibiting optimal enzyme activity at
55 °C whereas MtMetAP1c is fully active at 37 °C. The two isozymes
also differ in their specificity towards substrates and metal ion
cofactors. Further, Zhang et al.8 determined that the mRNA levels
for the two isoforms were vastly different in different growth
phases—mapA gene was expressed more in log phase, while mapB
transcript levels were higher in stationary phase—suggesting that
the two MetAPs are important for Mtb growth in different phases
of its unique life cycle. Using a combination of chemical and genetic
approaches, we had validated MtMetAPs to be viable targets. MtMe-
tAP1a had emerged as a more sensitive target in the culture setting
of Mtb.9 In an effort to identify novel inhibitors of MtMetAPs, we
conducted a high-throughput screen (HTS) of a 175,000 compound
library (from ASDI, Inc., Newark, DE) against MtMetAP1c. One of the
most potent hits against MtMetAP1c was N0-hydroxy-N-(7-meth-
MetAPs are metalloproteases that perform the vital function of
removing the initiator methionine from the nascent polypeptides
emanating from ribosomes, and hence are expressed and present
⇑
Corresponding author. Tel.: +1 410 955 4619; fax: +1 410 955 4620.
E-mail address: joliu@jhu.edu (J.O. Liu).
Current address: Department of Pharmaceutical Sciences, College of Pharmacy
and Health Sciences, Texas Southern University, Houston, TX 77004, USA.
à
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.022

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S. Bhat et al. / Bioorg. Med. Chem. 20 (2012) 4507–4513
oxy-4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide (4c, see
Table 1). This article is an account of our medicinal chemistry effort
at optimizing this hit for potent inhibition of both MtMetAPs.
3. Results and discussion
A target-based HTS of a 175,000 compound library from ASDI,
Inc., Newark, DE, was performed to identify novel inhibitors of
MtMetAP1c using a coupled enzyme assay20 with Met-Pro-pNA
as the substrate, Co2+ as the cofactor, and proline aminopeptidase
from Bacillus coagulans (BcProAP) as the coupling enzyme.8 The
HTS furnished us a hit bearing a novel N0-hydroxy-N-(7-meth-
oxy-4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide struc-
2. Chemistry
Compounds 3n through 3r were from the ASDI compound library
and they were verified by ESI-MS analysis to confirm the presence of
expected molecular ions. The N0-hydroxy-N-(thiazol-2-yl)methani-
midamides were prepared using a three-step protocol starting from
ketones 1 (Scheme 1). The ketones unavailable from commercial
sources, namely 1f,10 1g,11 1h,12 1j,13 and 1k14 were prepared fol-
lowing literature procedures. Aminothiazoles 2a–2m were pre-
ture, 4c with an IC50 of 1.33 lM. We then undertook a systematic
structure–activity relationship (SAR) study to fine–tune the struc-
ture for inhibition against MtMetAPs. As mentioned previously,
each of the isoforms, MtMetAP1a and MtMetAP1c, appears to be
important for TB during different growth phases, and hence, we as-
sayed all the compounds against both isoforms. Also, it is still un-
clear what the physiologically relevant metal cofactors are for
these two isoforms. In the in vitro assay setting, Wang’s group
demonstrated that highest catalytic activity of MtMetAP1a could
be achieved with Co2+ as the cofactor followed by Mg2+ and Mn2+
with nearly 2- and 3.5-fold less efficiency, respectively when com-
pared to Co2+ activation.8 On the other hand, Ye’s group reported
that the most efficient activators of MtMetAP1a were Ni2+ and
pared using a variation of Hantzsch thiazole synthesis,15 where
a-
halo ketones generated in situ (from 1a–1m) were reacted with
thiourea. Amide 3s was synthesized by coupling a silyl-protected
(S)-lactic acid with aminothiazole 2c and by subsequent removal
of the silyl protection revealing the free hydroxy group. The Schiff
bases 3t and 3v were made from 2c by treating them with furfural
and salicylaldehyde, respectively, following a reported procedure.16
The Schiff base 3t was reduced using sodium borohydride to form
the alkylated thiazole 3u. The aminothiazoles (2a–2m) were further
converted to the intermediates N0,N0-dimethylamidines 3a–3m by
refluxing with DMF–dimethylacetal. And finally, the formamidine
intermediates (3a–3m) were transformed into N0-hydroxy-N-(thia-
zol-2-yl)methanimidamides 4a–4m by treating with hydroxyl-
amine, as per the protocol of Sasaki et al.17 The overall yields of
4a–4m starting from the ketones (1a–1m) ranged from 37% to 75%.
N0-Hydroxy-N-(thiazol-2-yl)methanimidamide can exist as two
distinct tautomers as shown in Scheme 2. In the case of methani-
midamide 4c, we were able to discern by 2D-NOESY experiment
that the prevailing tautomer in acetone is 4c–2. This observation
is in complete agreement with the literature reports where N0-hy-
droxy-N-arylmethanimidamides were seen in this tautomeric form
in the X-ray structures.18,19
Co2+ ions (in that order).21 When it comes to MtMetAP1c, Co2+
Mn2+, Zn2+, and Mg2+ (in that order) were shown to be the effective
activators by Wang’s group;2 whereas Ye’s group established kcat
kM (in Mꢀ1, Sꢀ1) to be 659, 232, and 107 for Ni2+, Co2+, and Mn2+
,
/
,
respectively.22 Under our assay conditions, Co2+ and Mn2+ (in that
order) were the most effective metal ion cofactors for the MtMe-
tAPs and hence we chose to carry out all the in vitro enzyme inhi-
bition assays using these two metal ions. A likelihood of the
coupling enzyme (BcProAP) inhibition in the MtMetAP assay was
ruled out by performing an additional control experiment.
First we wanted to interrogate the importance of the N0-hydroxy-
methanimidamide functionality of the hit 4c, and thus we browsed
throughtheASDIlibraryand testedstructurallyrelatedanalogs. Nei-
Table 1
4H,5H-Naphtho[1,2-d]thiazole derivatives—MtMetAP assays
Note: IC50 values are the average of at least three independent experiments, each consisting of triplicates and standard deviation is given.

S. Bhat et al. / Bioorg. Med. Chem. 20 (2012) 4507–4513
4509
R1
X
R1
R1
A
R2
R3
R2
R3
R2
R3
Z = N=CHNMe2 (3a–3m)
vi
Z = NHCH=NOH (4a–4m)
O
i
ii-v
N
S
N
C
Z
NH2
B
Y
X
X
S
Y
Y
1
2
3
1, 2, 3, and 4: Substituents (when not specified, R#, X–Y = H)
Code
R1, R2, R3
X–Y
Z
–
3a – 3m, Z = N=CHNMe2
a
b
c
d
e
f
R1 = OMe
R2 = OMe
R3 = OMe
R1, R2 = OMe
R1–R2 = OCH2O
R2 = OMe
R1–R2 = OCH2O
R2 = OMe
4a – 4m, Z = NHCH=NOH
Other thiazole 3 derivatives: R1, R2, R3, and
CH2CH2
X–Y defined by a or b but differing in Z
3n = a, while Z = NH3+Br–
3o = b, while Z = NH3+Br–
3p = b, while Z = NHCOCH3
3q = a, while Z = NHCO(CH2)7CH3
g
h
i
OCH2
(RS)-OCH(Ph) 3r = b, while Z = NHCO(CH2)7CH3
(CH2)3
O(CH2)2
CH2
3s = b, while Z = (S)-NHCOCH(OH)CH3
3t = b, while Z = NH=CH(2-furanyl)
3u = b, while Z = NCH2(2-furanyl)
3v = b, while Z = N=CHPh(2-OH)
j
R1–R2 = OCH2O
k
l
R2 = OMe
R1, R2 = OMe
m
H (no ring)
Scheme 1. Synthesis of thiazole derivatives. Reagents and conditions: (i) I2/(NH2)2C@S, EtOH, 100 °C, 2–3 h; then free-base with NaHCO3; (ii) furfural or salicylaldehyde,
anhydrous MgSO4, 1:1 CH2Cl2–MeOH, rt, 6 h; (iii) (OMe)2CHNMe2/toluene, reflux, 4–6 h; (iv) (S)-(O)-TBDPS-lactic acid, HBTU/i-Pr2NEt, THF, 16 h, then n-Bu4NF/THF; (v) 3,
NaBH4/MeOH, rt, 16 h; (vi) NH2OHꢁHCl, MeOH, rt, 18 h.
nOe
9.42 (br, d)
MeO
MeO
9.38 (s)
ited both the metalloforms of MtMetAPs while having an IC50 of
1.26
M and 380 nM for Co2+-MtMetAP1a and Co2+-MtMetAP1c,
respectively.
To explore the SAR further about the B-ring, we tested a ring-
contracted version, indan derivative 4l, and a ring-excised version
4m. Of the two, 4l was not an inhibitor of MtMetAPs, but 4m did
inhibit MtMetAP1c with moderate potency, emphasizing the rele-
vance of B-ring, important perhaps in fixing the orientation of the
N0-hydroxymethanimidamide pharmacophore.
H
N
H
l
H
N
O
N
NH
OH
N
S
N
S
H
4c-1
4c-2
7.65
(d, JH,NH = 10 Hz)
Scheme 2. Tautomers of N0-hydroxymethanimidamides, example 4c–1 and 4c–2.
Evidence form NOESY suggesting a preponderence of 4c–2.
ther of the simple naphthothiazolyl amine salts 3n and 3o nor their
amide derivatives 3p, 3q, and 3r inhibited MtMetAPs (Table 1). At
Although N0-hydroxymethanimidamide moiety appears to be
intuitively acid labile, it has proved to be quite useful in improving
the oral bioavailability of methanimidamide containing drug
candidates, in particular as a prodrug approach23 (examples: anti-
thrombotic agent sibrafiban18 and a direct thrombin inhibitor
called ximelagatran24). Another salient example of an N0-hydroxy-
methanimidamide is an inhibitor of 20-hydroxyeicosatetraenoic
acid, called TS-011 which is being evaluated for the treatment of
cerebral ischaemia.25 Considering these successes, the MtMetAP
inhibitors we have described here are not likely to defy the
druggability criteria.
this point we reasoned that the presence of an oxygen atom in
a or
b position with respect to the nitrogen atom of aminothiazole might
be necessary for the inhibitory activity, and we prepared and tested
lactamide 3s, 2-furyl derivative 3u, and Schiff bases 3t and 3v. All the
analogs listed above failed to inhibit either MtMetAPs. Only meth-
animidamide 3a showed a weak inhibition of both MtMetAPs. Thus
we concluded N0-hydroxy-N-(thiazol-2-yl)methanimidamide to be
the optimal pharmacophore and focused our attention on varying
substitutions on the A-ring (see 3 in Scheme 1 for ring designation).
With examples 4a through 4f (see Table 2), we systematically
probed the effect of methoxy substitution on the A ring (4b, 4c,
and 4d), where compound 4b stood out for its good potency
against the two metalloforms of MtMetAPs. Dimethoxy derivative
4e was less potent against MtMetAPs and the methylenedioxy sub-
stitution (4f) led to even poorer inhibition. We then altered the B-
ring by replacing the benzylic methylene with an oxygen atom
(examples 4g, 4h, and 4i). A single methoxy group substitution
on the A-ring combined with or without an additional phenyl
group substitution on the B-ring (4g or 4i), produced compounds
exhibiting moderate inhibition of both the metalloforms of MtMe-
tAP1a, but they did not inhibit MtMetAP1c. Contrary to the case of
4f, methylenedioxy substituted compound 4h turned out to be po-
tent at inhibiting the cobalt-form of MtMetAP1c (730 nM) with
3.1. Metal complexation studies
We recorded UV–vis spectra of 4g, 4h, and 4k, in the MetAP assay
buffer (at 10 lM) with or without 10 lM cobalt or manganese chlo-
rides, which closely emulated the enzyme assay conditions. All
three compounds form metal complexes and yet despite such a close
structural relationship 4g did not inhibit either metalloforms of
MtMetAP1c. It should be noted that salicylaldimine like 3v is a
well-established metal ion chelator, but it did not inhibit MtMetAPs.
Thus, it appears that akin to our earlier experience with the human
MetAP inhibitors,26 ability to complex metal ions is not a sufficient
or necessary criterion for rationalizing MtMetAP inhibition.
good inhibition of MtMetAP1a as well (1.43
l
M, Co2+-MtMetAP1a).
3.2. Anti-tuberculosis activity
We then synthesized the B-ring expanded versions 4j and 4k, and
gratifyingly, 4k became the most potent MtMetAP inhibitor. Unlike
the benzosuberan congener 4j, benzoxepine derivative 4k inhib-
We determined the minimum inhibitory concentrations (MICs)
of methanimidamides 4a–4m in the culture of Mtb (H37Rv strain).

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S. Bhat et al. / Bioorg. Med. Chem. 20 (2012) 4507–4513
Table 2
Analogs of N0-Hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-yl)methanimidamide—MtMetAP assays and MICs (Mtb, H37Rv)
Entry
Structure
Substituents (when not specified, R# = H)
IC50
(l
M), MtMetAP
MIC (lg/mL)
1a, Co2+
1a, Mn2+
1c, Co2+
1c, Mn2+
R1
4a
4b
4c
4d
4e
4f
—
R
11.53 3.5
1.90 0.5
5.35 1.5
11.26 4.1
14.56 3.3
15.44 4.6
>50
>50
>50
11.67 3.2
14.6 3.6
>20
1.05 0.39
>50
2.33 0.39
>50
10.30 3.9
>50
100
200
>200
>200
>200
>200
1 = OMe
0.64 0.08
1.33 0.24
1.89 0.3
3.77 0.4
>20
R2
R2 = OMe
R3 = OMe
OH
N
N
S
R3
H
N
R1,R2 = OMe
R1–R2 = OCH2O
12.5 3.1
H
H
R1
4g
4h
4i
R2 = OMe
4.9 1.9
1.43 0.38
6.7 1.9
4.7 1.2
>10
9.7 2.3
>50
0.73 0.09
>50
>50
4.4 0.9
>50
>200
100
>200
R1–R2 = OCH2O
R2
R3
R2 = OMe; R = Ph
OH
N
N
S
H
N
O
R
O
4j
4k
X = CH2
X = O
15.4 4.1
1.26 0.2
>50
24.5 2.2
1.4 0.25
0.38 0.05
14.5 3.6
3.13 0.9
>200
>200
O
OH
N
H
N
N
X
S
H
OH
N
MeO
N
S
H
N
4l
—
—
>50
>20
>50
>20
>50
>50
100
200
H
MeO
MeO
4m
6.6 1.8
7.0 1.7
OH
N
N
H
N
S
H
O
O
Br
Br
NQa
—
1.14
ND
0.71
ND
25
Note: IC50 values are the average of at least three independent experiments, each consisting of triplicates and standard deviation is given.
a
Presented in our earlier work8 and is given here for comparison.
Except 4a, 4h, and 4l, which exhibited MICs of a meager 100 lg/
4. Conclusions
mL, the remaining compounds did not affect the growth of Mtb.
The positive control on the other hand, 2,3-dibromonaphthoqui-
none (NQ, IC50s, 1.14 [Co2+-MtMetAP1a], and 0.71 [Co2+-MtMe-
Starting from an N0-hydroxy-N-(naphthothiazolyl)methanimi-
damide 4c as a hit in an HTS, we established N0-hydroxymethani-
midamide as the required pharmacophore for the inhibition of
the two Mtb MetAP isoforms. Three inhibitors 4b, 4h, and 4k pos-
sessing potent activity against both MtMetAP1a and MtMetAP1c
were uncovered through an SAR campaign where benzoxepine
derivative 4k was identified as the best inhibitor. Unfortunately
the MtMetAP inhibitory potency of these methanimidamides did
not transpire into good MICs in Mtb culture. Undoubtedly, the
N0-hydroxymethanimidamide inhibitors need to be tuned further
with more SAR to find analogs that are effective in Mtb culture,
and the SAR described herein is an important step towards achiev-
ing that goal.
tAP1c) had an MIC of 25 l
g/mL matching our previous results.8
On the basis of enzyme inhibition data, we were expecting 4b,
4h, and 4k to manifest superior MICs, but counter to our expecta-
tions only 4h had a weak MIC of 100
damide 4l did not display any MtMetAP inhibitory activity, it
nevertheless registered an MIC of 100 g/mL. This result is quite
lg/mL. Although methanimi-
l
puzzling, because MICs did not correlate with the enzyme inhibi-
tion. In the MtMetAP target validation study we conducted previ-
ously, NQ was effective in blocking the Mtb growth. A plausible
interpretation of this puzzling result of MICs is that the methani-
midamides 4a–4m either lack the ability to permeate the bacterial
cell or they succumb to an intrinsic efflux mechanism.27,28 A con-
trol experiment was conducted to allay suspicion of instability of
N0-hydroxymethanimidamide functionality and unsurprisingly
compounds 4a–c and 4k were quite stable to the Mtb culturing
conditions (see Supplementary data). Curiously, an MtMetAP
inhibitor bengamide analog was reported to exhibit a reasonable
5. Experimental
5.1. Chemistry: general methods
Unless stated otherwise, all non-aqueous reactions were carried
out under ambient atmosphere in oven-dried glassware. Indicated
reaction temperatures refer to those of the reaction bath, while
inhibition of replicating Mtb (IC50s,
l
M: 6.9 [Mn2+-MtMetAP1a],
g/mL).29
0.54 [Mn2+-MtMetAP1c], MIC: 18.5
l

S. Bhat et al. / Bioorg. Med. Chem. 20 (2012) 4507–4513
4511
room temperature (rt) is noted as 25 °C. All the solvents were of re-
agent grade purchased from Fisher Scientific or VWR and used as
received. Commercially available starting materials and reagents
were purchased from Acros, Aldrich, or TCI America and were used
as received, unless stated otherwise.
Analytical thin layer chromatography (TLC) was performed
using Analtech Uniplates (silica gel HLF, W/UV254, 250 lm). Visu-
added to a 1:1 CH2Cl2–MeOH mixture containing anhydrous
MgSO4 (1 g) and the reaction mixture was stirred under argon for
6 h (the solution turned yellow after 30 min). The mixture was fil-
tered and the filtrate was concentrated. The previously known
product (3t) was purified by column chromatography over silica
gel (eluent: 1:9 EtOAc/hexanes). Yield: 404 mg, 94%; Rf (3:7
EtOAc/hexanes): 0.75 (UV active); 1H NMR (400 MHz, acetone-
d6): d, 7.97 (s, 1H, HC@N), 7.79 (d, J = 9.2 Hz, 1H, H-9), 7.35 (d,
J = 2.1 Hz,1H, H-50), 6.85 (m, 3H, H-6,8; H-30), 6.77 (m, 1H, H-40),
3.82 (s, 3H, OMe), 2.86–2.82 (m, 4H, H-4,5); MALDI-TOF: m/z,
311 (100%, MH+), 350 (15%, M+K+).
alization was achieved using 254 nm UV light, or additionally by
staining with iodine, or ceric ammonium molybdate stain. The
retention factor is reported as follows: Rf (eluent system). Crude
products were purified by air-flashed column chromatography
over silica gel (0.06–0.2 mm, 60 Å, from Acros) using indicated elu-
ents. Melting points were recorded on a Mel-Temp II apparatus and
are uncorrected. NMR data were collected on a Varian Unity-400
(400 MHz 1H, 100.6 MHz 13C) or Bruker-Spectrospin 400 MHz
spectrometers. Chemical shifts are reported in ppm (d) with the
solvent resonance or 0.1% tetramethylsilane contained in the deu-
terated solvent as the internal reference. Data are reported as fol-
lows: chemical shift, multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, dd = doublet of doublet, m = multiplet,
br = broad, app = apparent, exch = exchangeable), coupling con-
stants (J, reported in Hertz, Hz), and number of protons. Low reso-
lution mass spectra were acquired on a Thermo-Finnigan MAT, LCQ
Classic ESI-mass spectrometer or Voyager DE-STR, MALDI-TOF (Ap-
plied Biosystems) instruments. The MALDI-samples were prepared
by mixing the droplets of the sample solutions in chloroform or
methanol and 2,5-dihydroxybenzoic acid solution in acetone,
where the latter served as the matrix. Data are reported in the form
m/z (% abundance, ion).
The Schiff base 3t (20 mg, 64.5
lmol) was dissolved in MeOH
(8 mL) and NaBH4 (15 mg, 395 mol) was added and the mixture
l
was stirred overnight. After concentrating, the reaction mixture
was purified by flash column chromatography over silica gel
(1.5:8.5 EtOAc/hexanes). Yield: 29 mg, 96%; Rf (3:7 EtOAc/hex-
anes): 0.73 (UV active); tR = 12.400 min (98%); 1H NMR
(400 MHz, acetone-d6): d, 7.81 (d, J = 9.2 Hz, 1H, H-9), 7.34 (d,
J = 2.1 Hz,1H, H-50), 6.89 (m, 3H, H-6,8; H-30), 6.78 (m, 1H, H-40),
4.66 (br s, 1H, NH), 4.32 (s, 2H, CH2-furyl), 3.85 (s, 3H, OMe),
2.86–2.82 (m, 4H, H-4,5); d, MALDI-TOF: m/z, 313 (100%, MH+),
335 (20%, M+Na+).
5.1.3. Analogs of N0-hydroxy-N-(4H,5H-naphtho[1,2-d]thiazol-2-
yl)methanimidamide
These derivatives were prepared using a three-step protocol
starting from an appropriate ketone. As a representative example,
synthesis of methanimidamide 4b is described below.
Samples were analyzed for purity on a JASCO HPLC equipped
5.1.3.1. Synthesis of 8-methoxy-4H,5H-naphtho[1,2-d]thiazol-2-
with a Rainin Microsorb-MV C18 column (5
l
m, 4.6 ꢂ 100 mm).
amine (2b).
Naphthothiazole 2b was prepared using a known
The mobile phase set at a flow rate of 1 mL/min was programmed
for a gradient elution starting from a 75:20:5–10:75:15 mixture of
H2O–MeCN–MeOH over 10 min, and the mixture was held steady
at that ratio for six more minutes, and during the next minute it
was reverted back to the original ratio of 75:20:5, making each
run a total of 17 min long. Purity of final compounds was deter-
procedure.15 Briefly, thiourea (3.88 g, 51 mmol) and iodine (4.75 g,
18.7 mmol) were added to a solution of tetralone 1b (3 g, 17 mmol)
in absolute ethanol. The reaction mixture was heated at 100 °C in
an open vessel for 3 h, and in the end all the solvent was allowed
to evaporate. The residue was washed with ether (3ꢂ 15 mL),
dissolved in water (50 mL) and heated for 30 min and cooled.
The white solid was filtered, dried, and recrystallized from 9:1
EtOH–H2O and dried under vacuum to afford the hydroidide salt
of 2b. Upon a quick free-basing by washing with 5% NaOH and
evaporating the dichloromethane layer, the sample was character-
ized by 1H NMR and MALDI-TOF and it was carried over to the next
step.
mined to be >95%, using a 10 lL injection (approximately 1 mM
in acetonitrile) with quantitation by area under the curve (AUC)
at 254, 275, and 296 nm (JASCO Diode Array Detector). The reten-
tion time (tR) is reported in minutes with the AUC given in
parentheses.
5.1.1. (S)-2-Hydroxy-N-(7-methoxy-4H,5H-naphtho[1,2-
d]thiazol-2-yl)propanamide (3s)
5.1.3.2. Synthesis of N0-hydroxy-N-(8-methoxy-4H,5H-naph-
7-Methoxy-4H,5H-naphtho[1,2-d]thiazol-2-amine
365 mol) was coupled with (S)-O-(tert-butyldiphenylsilyl)lactic
acid30 using HBTU (152 mg, 1.1 equiv) and i-Pr2NEt (230
L,
(85 mg,
tho[1,2-d]thiazol-2-yl)methanimidamide (4b).
A two step
l
sequence described in the literature17 was applied to making
methanimidamide 4b. Aminothiazole 2b (1.4 mmol) from the pre-
vious step was heated at 100 °C with N,N-dimethyl-formamide di-
l
3.5 equiv) in THF (10 mL). After stirring the reaction mixture over-
night, water (10 mL) was added and the product was extracted into
EtOAc (2ꢂ 15 mL). The pooled organic phase was evaporated and
the product was diluted with THF (10 mL) and treated with n-
Bu4NF (1.2 mL, 1 M in THF) at room temperature for 4 h. The reac-
tion mixture was concentrated and the residue was subjected to
flash column chromatography over silica gel (eluent, 1:5 EtOAc/
hexanes) to yield an off-white solid. Yield: 59 mg, 53% (over two
steps); Rf (3:7 EtOAc/hexanes): 0.21 (UV active); tR = 9.080 min
(98%); 1H NMR (400 MHz, CDCl3): d, 7.55 (d, J = 9.1, 1H, H-9),
7.37 (br s, 1H, NH), 6.79 (m, 2H, H-6,7), 4.54 (m, 1H, H-10), 3.83
(s, 3H, OMe), 3.07–2.86 (m, 4H, H-4,5), 1.28 (d, J = 6.7 Hz, 3H, H-
30); MALDI-TOF: m/z, 305 (30%, MH+), 327 (80%, M+Na+).
methyl acetal (220 lL, 1.64 mmol) in toluene (15 mL) for 5 h and
the solvent was evaporated. The residue was recrystallized from
cyclohexane and the resulting solid (4b) was stirred with hydrox-
ylamine hydrochloride (575 mg, 8.3 mmol) in methanol (20 mL) at
room temperature for 16 h. The reaction mixture was concentrated
and it was neutralized by adding 10% Na2CO3 solution dropwise
(pH 9). A brown precipitate was formed and it was filtered and
washed with water and recrystallized further from 1,4-dioxane
to afford pure methanimidamide 4b.
5.1.3.3. Methanimidamide 4b.
Yield: 52% over three steps; Rf
(3:7 EtOAc/hexanes): 0.42 (UV active); tR = 8.013 min (98%); 1H
NMR (400 MHz, acetone-d6): d, 9.38 (br s, 2H, NH, OH), 9.26 (s,
1H, CH@N), 7.52 (d, J = 8.6 Hz, 1H, H-6), 7.37 (d, J = 1.9 Hz,1H, H-
9), 6.85 (dd, J = 8.6, 1.9 Hz, 1H, H-7), 3.75 (s, 3H, OMe), 3.11–2.92
(m, 4H, H-4,5); MALDI-TOF: m/z, 276 (35%, MH+), 298 (20%,
M+Na+).
5.1.2. N-(Furan-2-ylmethyl)-7-methoxy-4H,5H-naphtho[1,2-
d]thiazol-2-amine (3u)
7-Methoxy-4H,5H-naphtho[1,2-d]thiazol-2-amine
(325 mg,
1.4 mmol) and freshly distilled furfural (162 L, 1.7 mmol) were
l

4512
S. Bhat et al. / Bioorg. Med. Chem. 20 (2012) 4507–4513
5.1.3.4. Methanimidamide 4d.
Methanimidamide 4d was
1H, CH@N), 6.49 (s, 1H, H-10), 5.92 (s, 1H, H-7), 5.91 (d, J = 6 Hz,
2H, OCH2O), 4.28 (t, J = 5.6 Hz, 2H, H-5) 3.18 (t, J = 5.6 Hz, H-4);
MALDI-TOF: m/z, 306 (85%, MH+), 328 (50%, M+Na+).
prepared from the commercially available 5-mehtoxy-1-tetralone.
Final compound was purified by flash chromatography over silica
gel (eluent: 2% MeOH–DCM). Yield: 61% over 3 steps; Rf (3:7
EtOAc/hexanes): 0.33 (UV active); tR = 9.547 min (98%); 1H NMR
(400 MHz, acetone-d6): d, 9.18 (br s, 2H, NH, OH), 9.12 (s, 1H,
CH@N), 8.05 (d, J = 8.7 Hz, 1H, H-9), 7.38 (d, J = 8.7 Hz, 1H, H-8),
6.85 (d, J = 8.6 Hz, 1H, H-7), 3.79 (s, 3H, OMe), 3.21–2.97 (m, 4H,
H-4,5); MALDI-TOF: m/z, 276 (60%, MH+), 298 (40%, M+Na+).
5.1.3.12. Methanimidamide 4l.
This prepared from the com-
mercially available 5-mehtoxy-1-indanone. Yield: 71% over three
steps; Rf (3:7 EtOAc/hexanes): 0.34 (UV active); tR = 8.040 min
(97%); 1H NMR (400 MHz, DMSO-d6): d, 10.51 (br s, 1H, NH or
OH), 10.33 (s, 1H, CH@N), 7.63 (s, 1H, NH or OH), 7.40 (d,
J = 8.8 Hz, 1H, H-8), 7.18 (d, J = 1.4 Hz, 1H, H-5), 6.88 (d,
J = 8.8 Hz, 1H, H-6), 3.79 (s, 3H, OMe), 3.76 (d, J = 11.2 Hz, 2H, H-
4); MALDI-TOF: m/z, 262 (66%, MH+), 284 (20%, M+Na+).
5.1.3.5. Methanimidamide 4e.
This was prepared from the
commercially available 6,7-dimehtoxy-1-tetralone (1e). Yield:
48% over three steps; Rf (5% MeOH/DCM): 0.28 (UV active);
tR = 8.187 min (96%); 1H NMR (400 MHz, acetone-d6): d, 9.61 (br
s, 2H, NH, OH), 7.81 (s, 1H, CH@N), 7.39 (s, 1H, H-9), 6.83 (s, 1H,
H-6), 3.83 and 3.81 (2ꢂ s, 6H, OMe), 3.22–2.85 (m, 4H, H-4,5);
MALDI-TOF: m/z, 306 (20%, MH+), 328 (M+Na+), 290 (100%, M+ꢀO).
5.1.3.13. Methanimidamide 4m.
This was prepared from the
commercially available 3,4-methylenedioxyacetophenone. Yield:
75% over three steps; Rf (5% MeOH/DCM): 0.42 (UV active);
tR = 7.187 min (97%); 1H NMR (400 MHz, acetone-d6): d, 8.67 (br
s, 2H, NH and OH), 7.18 (s, 1H, CH@N), 6.91 (d, J = 8.8 Hz, 1H, H-
6), 6.62 (d, J = 2.4 Hz,1H, H-10), 6.41 (s, 1H, H-5), 6.03 (d,
J = 8.8 Hz, 1H, H-7), 2.94 and 2.91 (2ꢂ s, 6H, OMe); MALDI-TOF:
m/z, 280 (70%, MH+), 263 (MH+ꢀOH).
5.1.3.6. Methanimidamide 4f.
This was prepared from 6,7-
(methylenedioxy)-1-tetralone (1f).10 Yield: 67% over three steps;
Rf (3:7 EtOAc/hexanes): 0.39 (UV active); tR = 9.507 min (97%); 1H
NMR (400 MHz, acetone-d6): d, 9.55 (br s, 2H, NH, OH), 7.89 (s,
1H, CH@N), 7.25 (s, 1H, H-9), 6.85 (s, 1H, H-6), 6.02 (d,
J = 10.3 Hz, 2H, OCH2O), 3.21–2.95 (m, 4H, H-4,5); MALDI-TOF:
m/z, 290 (90%, MH+), 312 (30%, M+Na+).
5.2. Biological assays
5.2.1. MetAP enzyme assays
The protein expression and purification of C-terminal 6ꢂHis-
tagged MtMetAP1a and N-terminal 6ꢂHis-tagged MtMetAP1c
were carried out as described before. HTS was also conducted as
was described in our earlier work.9 However the MtMetAP assays
in the present study were performed with slight modification as
explained underneath.
5.1.3.7. Methanimidamide 4g.
This was prepared from 7-
methoxy-2H,3H,4H-chromen-4-one.11 Yield: 69% over three steps;
Rf (3:7 EtOAc/hexanes): 0.43 (UV active); tR = 8.943 min (97%); 1H
NMR (400 MHz, acetone-d6): d 9.51 and 9.48 (2ꢂ br s, 2H, NH
and OH), 9.36 (s, 1H, CH@N), 7.65 (d, J = 9.2 Hz, 1H, H-9), 7.57 (d,
J = 9.2, Hz,1H, H-8), 6.70 (d, J = 6 Hz, 1H, H-6), 5.35 (d, J = 9.6 Hz,
2H, H-4); MALDI-TOF: m/z, 278 (76%, MH+), 300 (55%, M+Na+).
Eleven different concentrations of naphthothiazole derivatives
(50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, 0.098,
0.049
bated for 20 min at room temperature with an assay buffer (40 mM
HEPES [pH 7.5], 100 mM NaCl and 10 M CoCl2 or MnCl2). The as-
say buffer also contained either 200 nM MtMetAP1a or 100 nM
MtMetAP1c, and 1.5 U/mL proline aminopeptidase (from Bacillus
coagulans, BcProAP) as the coupling enzyme.31 After 20 min prein-
cubation with the compounds, the enzyme reaction was initiated
lM) were pipetted into a 96-well plate and they were incu-
5.1.3.8. Methanimidamide 4h.
Methanimidamide 4h was
prepared from 6,7-dihydro-8H-1,3-dioxolo[4,5-g]benzopyran-8-
one.12 Yield: 55% over three steps; Rf (3:7 EtOAc/hexanes): 0.36
(UV active); tR = 9.320 min (96%); 1H NMR (400 MHz, acetone-
d6): d 9.64 and 9.55 (2ꢂ br s, 2H, NH and OH), 7.79 (s, 1H, CH@N),
7.17 (s, 1H, H-9), 6.51 (s, 1H, H-6), 6.02 (d, J = 10.6 Hz, 2H, OCH2O),
5.33 (d, J = 9.8 Hz, 2H, H-4); MALDI-TOF: m/z, 292 (80%, MH+), 314
(45%, M+Na+).
l
by adding methionylprolyl-p-nitroanilide as the substrate (20
3 mM; such that a final concentration of 600 M was attained in
a 100 L of total reaction volume in each well) and the change in
lL,
l
l
5.1.3.9. Methanimidamide 4i.
This was prepared from the
absorbance at 405 nm was measured using a microplate reader
(BMG Labtech, Offenburg, Germany) for a period of 20 min. Reac-
tion progress was computed against the data from the wells con-
taining DMSO alone taken for uninhibited reactions. In the
control experiments to rule out the possibility of the inhibition of
BcProAP itself, the assay protocol was modified where prolyl-p-
nitroanilide was used as the substrate (obviously MtMetAP was
eliminated). EC50 values were determined by plotting the data
using nonlinear regression analysis in the GraphPad Prism 4.0 (La
Jolla, CA) and the data from at least three independent experiments
(each consisting of a triplicate) and IC50s reported are the average
while the error indicated is the standard deviation.
commercially available 7-methoxyflavanone. Yield: 61% over three
steps; Rf (3:7 EtOAc/hexanes): 0.54 (UV active); tR = 10.403 min
(97%); 1H NMR (400 MHz, methanol-d4): d, 8.31 (br s, 2H, NH
and OH, partially exchanged), 7.75 (dd, J = 8.8, 1.4 Hz, 1H, H-9),
7.52–7.33 (m, 6H, CH@N, phenyl), 6.55 (dd, J = 8.8 and 2.4 Hz,
1H, H-8), 6.49 (dd, J = 2.4 and 1.4 Hz, 1H, H-6), 5.08 (s, 1H, H-4),
3.78 (s, 3H, OMe); MALDI-TOF: m/z, 354 (65%, MH+), 376 (40%,
M+Na+).
5.1.3.10. Methanimidamide 4j.
from 6,7,8,9-tetrahydro-5H-cyclohepta[f]-1,3-benzodioxol-5-
This compound was prepared
one.13 Yield: 37% over three steps; Rf (3:7 EtOAc/hexanes): 0.45
(UV active); tR = 9.987 min (98%); 1H NMR (400 MHz, acetone-
d6): d, 9.57 (br s, 2H, NH, OH), 7.90 (s, 1H, CH@N), 7.27 (s, 1H, H-
10), 6.88 (s, 1H, H-7), 6.05 (d, J = 10.3 Hz, 2H, OCH2O), 3.11–2.85
(m, 4H, H-4,6), 2.28 (m, 2H, H-5); MALDI-TOF: m/z, 304 (70%,
MH+), 326 (40%, M+Na+).
5.2.2. Determination of MICs
Compound concentrations prepared directly in the medium
were 0, 0.8, 1.6, 3.2, 6.3, 12.5, 25, 50, 100, 200 and 400
lg/mL. A
100 L volume of Middlebrook 7H9 broth (Difco, USA) with com-
l
pound in different concentrations was dispensed into each well
of a 96-well cell culture plate (BD). A standard log-phase growth
bacterial suspension (Mycobacterium tuberculosis, H37Rv strain)
was prepared and diluted to ꢃ106 CFU/mL in 7H9 broth and a
5.1.3.11. Methanimidamide 4k.
This was prepared from
7H,8H-1,3-dioxolo[4,5-h]benzoxepin-9(6H)-one.14 Yield: 51% over
three steps; Rf (3:7 EtOAc/hexanes): 0.32 (UV active);
tR = 8.640 min (96%); 1H NMR (400 MHz, methanol-d4): d, 7.69 (s,
100 lL inoculum was used to inoculate each well of the plate. A
sterile control without inoculum was also included. Plates were

S. Bhat et al. / Bioorg. Med. Chem. 20 (2012) 4507–4513
4513
9. Olaleye, O.; Raghunand, T. R.; Bhat, S.; He, J.; Tyagi, S.; Lamichhane, G.; Gu, P.;
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the minimum compound concentration at which the well was clear
indicating that no bacterial growth had occurred.
11. Vu, A. T.; Campbell, A. N.; Harris, H. A.; Unwalla, R. J.; Manas, E. S.; Mewshaw, R.
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This work was supported in part by the Department of Pharma-
cology, Johns Hopkins School of Medicine and the Keck Foundation.
We are grateful to ASDI, Inc. for the provision of the compound li-
brary. We thank Mr. Steve Fernandes for technical assistance.
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Supplementary data
Supplementary data (these data include representative MtMe-
tAP inhibition curves, representative UV–vis spectra recorded as
part of the metal complexation studies, and HPLC chromatograms
attesting to the stability of N0-hydroxymethanimidamides (4a, 4b,
4c, and 4k) under Mtb culturing conditions) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2012.05.022.
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