Thiadiazole carbamates: Potent inhibitors of lysosomal acid lipase and potential niemann-pick type C disease therapeutics

Article abstract of DOI:10.1021/jm100499s

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles. Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates. An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that furnishes the products in high yields and high degrees of purity. Common intermediates can also be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a diverse selection of commercially available non-thiadiazole carbamates. Our studies show that, among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by rivastigmine and other carbamates as well as acylation of various lipases by orlistat.

Full text of DOI:10.1021/jm100499s

NIH Public Access
Author Manuscript
J Med Chem. Author manuscript; available in PMC 2011 July 22.
Published in final edited form as:
J Med Chem. 2010 July 22; 53(14): 5281–5289. doi:10.1021/jm100499s.
Thiadiazole Carbamates: Potent Inhibitors of Lysosomal Acid
Lipase and Potential Niemann-Pick Type C Disease
Therapeuticsa
#,†
#,‡
‡
Anton I. Rosenbaum , Casey C. Cosner , Christopher J. Mariani , Frederick R.
†,*
‡
‡,*
Maxfield , Olaf Wiest , and Paul Helquist
†Department of Biochemistry, Weill Cornell Medical College, 1300 York Avenue, New York, NY
10065
‡Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science
Hall, Notre Dame, IN 46556
Abstract
Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized at the cellular
level by abnormal accumulation of cholesterol and other lipids in lysosomal storage organelles.
Lysosomal acid lipase (LAL) has been recently identified as a potential therapeutic target for
NPC. LAL can be specifically inhibited by a variety of 3,4-disubstituted thiadiazole carbamates.
An efficient synthesis of the C(3) oxygenated/C(4) aminated analogues has been developed that
furnishes the products in high yields and high degrees of purity. Common intermediates can also
be used for the synthesis of the C(3) carbon substituted derivatives. Herein we tested various
thiadiazole carbamates, amides, esters, and ketones for inhibition of LAL. In addition, we tested a
diverse selection of commercially available non-thiadiazole carbamates. Our studies show that,
among the compounds examined herein, only thiadiazole carbamates are effective inhibitors of
LAL. We present a mechanism for LAL inhibition by these compounds whereby LAL transiently
carbamoylates the enzyme similarly to previously described inhibition of acetylcholinesterase by
rivastigmine and other carbamates as well as acylation of various lipases by orlistat.
Introduction
Niemann-Pick type C disease (NPC)1 is a rare, incurable, autosomal recessive lysosomal
storage disorder.2 The disease is characterized by significant accumulation of unesterified
cholesterol, glycosphingolipids, and other lipids within late endosomes/lysosomes (LE/LY).
3 Clinical manifestations include liver abnormalities, epilepsy, seizures, and significant
neurodegeneration, ultimately resulting in fatal outcomes. While miglustat4 and
a
Abbreviations: ACAT, acyl Co-A: cholesteryl acyl transferase; CE, LDL-associated cholesterol esters; DMAP, 4-
dimethylaminopyridine; EDC, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride; LAL, lysosomal acid lipase; LDA,
lithium diisopropyl amide; LE/LY, late endosomes/lysosomes; LiHMDS, lithium hexamethyldisilazide; LSO, lysosomal storage
organelle; NPC, Niemann-Pick type C disease; phLAL, purified human lysosomal acid lipase; SAR, structure-activity relationship;
TFA, trifluoroacetic acid.
*Corresponding authors: Frederick R. Maxfield frmaxfie@med.cornell.edu Department of Biochemistry Weill Cornell Medical
College 1300 York Avenue New York, NY 10065 Tel: (212)-746-6405 Fax: 212-746-8875. *Paul Helquist phelquis@nd.edu
Department of Chemistry and Biochemistry 251 Nieuwland Science Hall University of Notre Dame Notre Dame, IN 46556 Tel:
(574)-631-7822 Fax: (574)-631-6652.
#
These authors contributed equally to this work.
Supporting Information: Detailed experimental procedures and characterization data for all intermediates, products, and procedures for
biochemical assays are available. This material is provided free of charge from http://pubs.acs.org.

Rosenbaum et al.
Page 2
cyclodextrin5 have become candidates for potential therapy, there remains a significant need
to find alternative small molecules for treating NPC disease.
In normal cells, LDL-associated cholesterol esters (CE) are transported via receptor-
mediated endocytosis to the LE/LY. Within this compartment, lysosomal acid lipase (LAL)
hydrolyzes CE to free cholesterol and fatty acids.6 Free cholesterol is then transported from
the LE/LY to various organelles, including the endoplasmic reticulum (ER) and the plasma
membrane.7
LDL receptor-mediated cholesterol uptake, transport to the LE/LY, and cholesteryl ester
hydrolysis by LAL are unaltered in NPC-deficient cells. However, the egress of liberated
cholesterol from the LE/LY is significantly reduced, causing them to become lysosomal
storage organelles (LSO's).8 Consequently, re-esterification by acyl Co-A:cholesteryl acyl
transferase (ACAT) is reduced.
It is not clear what aspect of defective cholesterol transport leads to neuronal cell death. One
possibility is that the accumulation of cholesterol and other lipids in the LSO's contributes to
the pathology. One possible therapeutic option for NPC patients is the reduction of
cholesterol within the LSO's. Treatment of NPC1- or NPC2-defective mice with β-
cyclodextrins causes a significant reduction in the accumulation of cholesterol and other
,
lipids in LSOs and significantly extends lifespan.5a 9 These results indicate that cholesterol
reduction may be a viable therapeutic option for NPC disease.
We recently presented the results of an automated filipin-based cellular assay that measures
the cholesterol levels within the LSO's.10 The assay is amenable to high throughput
screening, allowing libraries of compounds to be investigated for the effect of cholesterol
reduction. The technique has already demonstrated some success with our findings that
certain pyrrolinones increased cholesterol esterification and decreased LDL uptake in NPC-
deficient cells.11 While these initial results are encouraging, the screening process simply
identifies compounds that revert the NPC phenotype in terms of cholesterol accumulation.
However, it does not identify the molecular target(s) affected by the compounds. An
analysis of cellular mechanisms related to cellular cholesterol homeostasis shows that a
decrease in cholesterol content within the LSO's can be explained by one or a combination
of the following mechanisms: 1) increase in cholesterol efflux from the LSO; 2) decreasing
the uptake of cholesterol; or 3) decrease in hydrolysis of cholesteryl esters by LAL.
Recently, thiadiazoles containing a carbamate moiety at C(3) (vide infra) were identified as
effective compounds in reducing the LSO cholesterol content using our fluorescence-based
assay.12 The cholesterol reduction was found to be a direct result of LAL inhibition. This
inhibitory activity is similar to orlistat, a well-documented inhibitor of several lipases
(Figure 1), which was also found as a hit in our screen.13 The IC values for the hit
50
compounds against purified human LAL (phLAL) were in the mid-nanomolar range. In
addition to the potent inhibition of LAL, the thiadiazoles were found to be selective for
LAL, exhibiting no inhibition of human pancreatic lipase or bovine milk lipoprotein lipase
at the concentrations tested. In contrast, orlistat is a potent inhibitor of many lipases.12
Furthermore, no apparent toxicity was observed for these thiadiazoles when cell viability
assays were conducted at 10 μM. The combined cholesterol reduction, selective inhibition of
LAL, and low toxicity of the thiadiazoles render them attractive candidates for further study
as potential NPC therapeutic agents. This paper describes the next logical phase of these
studies, including development of improved synthetic methods of broader scope for this new
class of inhibitors, establishment of structural requirements for activity, and more detailed
enzyme inhibition and cell studies.
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Page 3
Chemistry
Previous syntheses of the 1,2,5-thiadiazole core are based upon two very different strategies.
14 The first strategy begins with α-amino nitriles, which are cyclized to the corresponding 3-
chloro-4-alkyl-1,2,5-thiadiazole using sulfur monochloride. This strategy has most notably
been used in the synthesis of xanomeline, an alkoxythiadiazole functioning as an
acetylcholine receptor agonist.15 While this is a common method for the synthesis of 1,2,5-
thiadiazoles, several drawbacks exist. First, it uses the highly toxic sulfur monochloride to
conduct the cyclization. Additionally, the yields are often low, and the starting α-amino
nitriles must be synthesized prior to their use in this approach. The second strategy begins
with commercially available 3,4-dichloro-1,2,5-thiadiazole 1. The halogens may be
displaced sequentially with various nucleophiles, furnishing unsymmetrical thiadiazole
products. This second strategy avoids the difficulties of the first pathway, and we
consequently adapted this route for our purposes.
The synthetic route for the initial hit compounds (from this point forward, referred to as C(3)
carbamates) is shown in Scheme 1. Beginning with commercially available 1, a single
nucleophilic substitution with a secondary amine provided the intermediates 2–5 in high
yield. Treatment with KOH afforded the hydroxy intermediates 6–9. The final step was the
acylation of the alcohols with the corresponding aminocarbonyl chloride to afford the C(3)
carbamates 10–17 (Table 1).
Some time dependence was previously observed in the LAL inhibition by thiadiazole
carbamates.12 An increase in inhibition was observed when the enzyme was pre-incubated
with the inhibitors for two hours as opposed to five minutes prior to the assay. This time
dependence may have resulted from the carbamoylation of the active LAL serine residue.
,
Previously, serine carbamoylation of acetylcholinesterase16 17 and butyrylcholinesterase18
has been shown to be an effective means of enzyme inhibition. To test this hypothesis, we
sought to synthesize analogues, which were structurally similar to 10–17, but would be
unable to participate in the carbamoylation pathway. Our synthetic route provided a unique
opportunity to provide the appropriate analogues readily by replacing the C(3)-oxygen with
a carbon. The resulting analogues, including amides, esters, and ketones, would be far less
labile than the carbamates, and therefore less prone to participate in the carbamoylation of
the LAL active serine residue. This strategy required that we use one of our intermediates 4
in reactions with carbon-based nucleophiles, specifically various types of enolates, to
synthesize the desired analogues. This need for the use of a variety of enolates provided a
good opportunity for development of synthetic methods producing a broader range of
thiadiazole derivatives than were previously accessible by simple, direct means.
While it has been reported that potent nucleophiles (Grignard reagents,19 alkyl lithium
reagents20) react with 1 at the sulfur atom and fragment the ring, there is lack of methods
based upon enolates reacting directly with monosubstituted thiadiazoles such as 2–5.
Consequently, we set out to find optimal conditions with which to conduct the substitution,
using thiadiazole 4 as the model substrate with ester enolates. After a significant amount of
experimentation, it was determined that using 2.3 equiv of LiHMDS and a slight excess of
the corresponding ester provided the desired substituted products in moderate to high yields
(Table 2). With this optimized procedure, there was no need to preform the ester enolate, but
rather the ester could be added to a solution containing both the thiadiazole and the base.
Generally, higher yields of the substitution products were obtained when sterically hindered
esters were employed (compounds 18, 21, and 26) versus smaller esters (compounds 19 and
20). Indeed, a common byproduct was the formation of the self-Claisen product, which
occurred more readily when sterically accessible esters such as ethyl acetate and methyl
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Rosenbaum et al.
Page 4
propionate were used. These byproducts were not observed when tert-butyl acetate and
methyl isobutyrate were employed, which afforded 18 and 21, respectively.
In addition to esters, ketone enolates also performed well. Aldol byproducts were often
observed under the standard conditions, but these could be avoided by quantitatively
forming the lithium enolates separately with LDA, and then adding the enolate solution to a
separate solution containing the thiadiazole and LiHMDS. By using this modified protocol,
acetophenone and propiophenone reacted well and provided 23 and 24, respectively, in good
yields. As with the sterically hindered esters, pinacolone could be employed using the
standard conditions, providing ketone product 25. Aldol byproducts were not observed with
pinacolone, a result consistent with the sterically hindered esters. Interestingly,
isobutyrophenone failed to react under both the standard conditions and the modified
protocol using the preformed enolate.
Although the corresponding amide analogues could in principle be obtained by an analogous
use of amide enolates, the readily obtained ester products are easily manipulated to provide
the desired amides. Two cases that we investigated were the tert-butyl acetate analogues 18
and 26. These esters were treated with TFA in CH Cl to furnish the free acids, which were
2
2
immediately treated with morpholine and EDC to supply the desired amides 27 and 28
(Scheme 2).
We had assumed that the enolate substitution was occurring through a direct nucleophilic
attack at C(3) to displace the chloride via a S Ar mechanism. However, Merschaert recently
N
reported that 1 cleanly reacts with LiHMDS at the sulfur atom to give a stabilized ring-
opened product.21 The resulting intermediate was then able to undergo substitution
reactions with various nucleophiles, including enolates. Given the similarities between our
optimal conditions and those reported for the ring opening, we investigated the possibility of
the reaction proceeding through an analogous ring opened intermediate.
When 4 was treated with 1.0 equiv of LiHMDS in THF at 22 °C, the starting material was
13
consumed within minutes (Scheme 3). C NMR analysis of the product 29 (isolated by
column chromatography) revealed a distinct shift in the carbon atoms of the thiadiazole ring
which matched those of an identical compound reported by Merschaert.21 When 29 was
treated with the Li enolate of tert-butyl acetate, the desired substitution product 18 was
isolated in good yield, albeit in lower yield than in the one-pot protocol.
Based on the above data, it is not likely that a direct nucleophilic substitution occurs at C(3)
of the thiadiazole ring. Rather, the substitution more likely occurs through a LiHMDS ring-
opened intermediate generated in situ, which undergoes facile reaction with enolate
nucleophiles and regenerates the thiadiazole ring.
Biological Activity
Our previous studies have identified several LAL inhibitors as potential therapeutics for
NPC disease.12 Among these LAL inhibitors were three thiadiazole carbamates: compounds
13, 14, 15 (named as 3a2, 3a7 and 3a6, respectively, in the previous publication), which
showed significant potency and specificity towards LAL. In this follow up study, we have
examined the structure-activity relationship (SAR) of these thiadiazoles and their derivatives
in terms of their ability to inhibit LAL in vitro and in cultured cells.
Enzyme kinetics analysis
In order to determine the type of inhibition of LAL by thiadiazole carbamates, we performed
a Lineweaver-Burk analysis for 13 using purified human LAL (phLAL). As shown in Figure
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Rosenbaum et al.
Page 5
2, compound 13 acts as an apparent competitive inhibitor. Since other compounds examined
herein are close structural analogues of 13 it would be reasonable to assume that their mode
of inhibition is also competitive.
In vitro activity assay and IC50 determinations
We have examined alterations of the compound structure that involve changes to both the
ring system at C4 and the specific groups located at C(3) of the thiadiazole ring. Only the
carbamate analogues (10–17) were found to be effective inhibitors. Their apparent IC 's, as
50
determined by an in vitro LAL activity assay, lie in the range of approximately 70 to 500
nM, as summarized in Table 1. Only one ester analogue (20) showed some limited efficacy
in inhibiting LAL, with an apparent IC =13 ± 2 μM. None of the ketone analogues and
50
neither of the two amides tested showed any inhibition of LAL activity at 10 μM, as shown
in Supplemental Figure 1. Thus, it appears that the carbamate moiety is required for
inhibition of LAL by thiadiazoles. In an attempt to test whether any carbamate would inhibit
LAL, we acquired a series of commercially available carbamates (see the Supporting
Information for the structures) of various degrees of complexity (methyl carbamate; guaiacol
glyceryl ether carbamate; 1-methyl-4-piperidinyl- N-(4-fluorophenyl) carbamate; benzyl N-
(6-aminohexyl) carbamate hydrochloride; eserine; neostigmine methyl sulfate). None of
these carbamates inhibited LAL at 10 μM, as shown in Supplemental Figure 2.
It appears that piperidine is the preferred substituent at the C4 position and morpholine at the
C(3) carbamate (13). Also an increase of the C4 substituent ring size from piperidine to
azepane was better tolerated than a decrease in ring size to pyrrolidine or a change to
morpholine (compare changes in IC 's between 13 and 16, 11 or 15). On the other hand, the
50
C(3) substituent ring seemed to be less sensitive to modifications in this in vitro assay as
both piperidine and morpholine substituents seemed equally tolerated (compare compounds
14 and 15).
Time-dependence of inhibition
We previously demonstrated that 13, 14, and 15 showed some time-dependence of inhibition
based upon pre-incubation of compounds with phLAL prior to reaction.12 As shown in
Figure 3, we observed similar pre-incubation time-dependence for all of the compounds
tested, except for 10, which showed significantly higher time-dependence than other
compounds. This slow time-dependence is in agreement with the hypothesis that LAL
inhibition is associated with serine carbamoylation.
Compound efficacy in cells
In order to examine compound efficacy in inhibiting LAL in cells, we measured enzyme
activity in lysates of cells that were treated for 4 days with thiadiazole carbamates at various
concentrations (Figure 4A). We observed that the compounds segregated into two distinct
groups based on the level of residual inhibition, i.e., 10, 12, 14, and 16 showed much higher
level of inhibition of LAL upon cell lysis than 11, 13, 15, and 17. The two groups of
compounds vary primarily in the nature of the C(3) substituent: a morpholine (10, 12, 14,
16) or piperidine (11, 13, 15, 17), which is expected to carbamoylate the catalytic serine of
LAL. Since the volume of a cell monolayer is much smaller than that of our reaction mixture
upon cell lysis, we postulated that the differences in inhibition observed was a consequence
of the differences in the apparent decarbamoylation rate of LAL modified by these
compounds upon the dilution of the enzyme with reaction buffer. We also observed that the
compounds that had a piperidine C(3) substituent inhibited LAL significantly better in cells
upon withdrawal of the compounds from the growth medium for 1 day after 3 days of
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Rosenbaum et al.
Page 6
treatment (Figure 4B). The compounds that had both the C(3) and the C4 substituent non-
oxygenated rings (12,16 and 10) exhibited maximal potency in this withdrawal assay.
LAL decarbamoylation rate determination
In order to test whether indeed the difference in inhibition between the morpholine and
piperidine thiadiazole carbamates observed was the result of different decarbamoylation
rates of the modified enzyme for the two groups, we examined enzyme reactivation
(decarbamoylation) in vitro under our standard reaction conditions (37 °C, 200 mM sodium
acetate pH 5.5, 0.02% Tween, 1.6 % Triton X-100, final) using phLAL. We pre-incubated
phLAL with compounds at high concentration (10 μM) to carbamoylate most of the enzyme
and then extensively diluted the enzyme (250X) with substrate and monitored the reaction at
10 min intervals for 2 hours at 37 °C. The decarbamoylation rates listed in Table 1 were
calculated as described previously.16 Indeed, we observed that the C(3) thiadiazole
morpholine carbamates 11, 13, 15, and 17 decarbamoylated several fold faster than their
piperidine analogues (10, 12, 14, 16).
In order to further test LAL inhibition in intact cells, we measured the decrease in
cholesterol accumulation in NPC1 mutant cells (Figure 4C) and the increase in neutral lipid
accumulation in normal cells (Figure 4D). It appears that 12 is the most potent thiadiazole
carbamate eliciting these effects, while 15 was least potent in all four assays, with the rest of
compounds exhibiting various degrees of efficacy.
Discussion and Conclusions
In summary, we have devised an efficient route for the synthesis of unsymmetrical 3,4-
disubstituted-1,2,5-thiadiazoles beginning with the commercially available 3,4-
dichloro-1,2,5-thiadiazole 1. The route produces C(3) oxygen-bonded carbamates in high
yields, is quite general, and is amenable to large scale work. Additionally, we have found
that C(3) carbon substituted thiadiazoles can be obtained by a simple one-pot substitution
with either ester or ketone enolates without isolation of the intermediates as reported
previously. The enolate reaction takes place rapidly and provides good to excellent yields of
the substituted products. Given the importance of the 1,2,5-thiadiazole core as a scaffold for
,
drug development,14 22 the new enolate-based transformation reported in this paper opens
up a wide range of new possibilities for the use of this heterocycle in medicinal chemistry.
The specific thiadiazole analogues synthesized exhibited a strong SAR in terms of LAL
inhibition in vitro and in cultured cells. Of the compounds tested herein, only the thiadiazole
carbamates were effective inhibitors. Only one ester (20) exhibited some low inhibitory
activity in vitro, and none of the amides or ketones studied showed activity at the
concentrations tested. Moreover, none of the commercially acquired non-thiadiazole
carbamates were effective in inhibiting LAL at the concentrations tested (Suppl. Fig. 2).
These findings point to the dual requirement of both the carbamate and the 3,4-disubstituted
thiadiazole moieties for successful inhibition of LAL. We also observed that thiadiazole
carbamates were competitive inhibitors of LAL (Figure 2) and exhibited time-dependence in
their ability to inhibit the enzyme (Figure 3). Thus, our data point to LAL carbamoylation as
the mechanism of inhibition by thiadiazole carbamates. Carbamate inhibitors of enzymes
,
such as acetylcholinesterase have been previously characterized.16 17 These inhibitors have
been shown to transiently carbamoylate the catalytic serine of acetylcholinesterase. A
similar mode of inhibition of LAL can be envisaged for the thiadiazole carbamates
examined in this study. The proposed mechanism of the modification of LAL by thiadiazole
carbamates is presented in Scheme 4.
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Rosenbaum et al.
Page 7
In this mechanism, the apparent efficacy of the compound would be determined by the three
reaction rates: the reversible rates of the binding of the enzyme to the compound (k , k ),
1
−1
the enzyme carbamoylation rate (k ) and the rate of decarbamoylation of the modified LAL
2
(k ). The rates k and k combine into the apparent, observable, inhibition rate k . Previous
3
1
2
i
work has demonstrated that the compound IC is related to k /k , under steady state
50
i
3
conditions.23
In order to explore the SAR of the thiadiazole carbamates' inhibition of LAL, we examined
their ability to inhibit LAL in vitro using purified enzyme and in cultured cells. Based on the
IC data summarized in Table 1, the best compound for in vitro inhibition of phLAL was
50
13, followed by 12. However, in the cellular context we observed that 12 was much more
potent in inhibiting LAL than 13. This was shown by the significant changes in the dose-
dependent decrease in cholesterol accumulation in NPC-mutant cells as well as the increased
neutral lipid accumulation in normal fibroblasts treated with these compounds. We
hypothesize that the difference in the time-scales of the two different sets of experiments
(i.e., in vitro vs. in cultured cells) was important in determining the apparent compound
efficacy. The in vitro experiments were usually conducted for a few hours or less (e.g., 90
min for IC determination), whereas the tissue culture experiments were conducted for
50
several days (e. g., 3 days for filipin/LipidTOX experiments). As outlined in Scheme 4, the
modified (and thus inhibited) enzyme will recover based upon the magnitude of the
decarbamoylation rate (k ). We observed strong preference for morpholine (11, 13, 15, 17)
3
as opposed to piperidine modification (10, 12, 14, 16) as shown in Table 1 in regards to the
ability of the enzyme to spontaneously recover after carbamoylation. This preference
appeared to affect compound efficacy in cells since this recovery rate would significantly
affect the more prolonged cell experiments, as opposed to the relatively short in vitro ones.
Compound 12 is a bispiperidine thiadiazole carbamate. The C(3) piperidine carbamate
moiety, after its attachment to the enzyme, would decarbamoylate at a significantly slower
rate than its morpholine analogue (13) and thus would be a major contributing factor to this
compound's increased potency in cells. The difference in the relative rates of
decarbamoylation is consistent with the difference in pK values of piperidine and
a
morpholine, the latter being significantly lower.24
In conclusion, we have explored the SAR of thiadiazole derivatives for LAL inhibition in
vitro and in cultured cells leading to further optimization of compound efficacy in a cellular
context and to significant elucidation of the molecular mechanism of action for this series of
compounds. Further structural studies would be needed to elucidate the precise mechanism
of LAL inhibition by these novel inhibitors as well as in vivo studies for the determination of
their pharmacokinetics and pharmacodynamics.
Experimental Section
Unless otherwise stated, all synthetic reactions were conducted under an inert atmosphere of
either nitrogen or argon. Reactions were monitored using thin layer chromatography (TLC)
performed on EMD plates. THF was distilled from sodium/benzophenone prior to use.
Methylene chloride (DCM) was distilled from CaH prior to use. 3,4-Dichloro-1,2,5-
2
thiadiazole was purchased from Alfa Aesar and used as received. MEM and LipidTOX
green were from Invitrogen (Carlsbad, CA). All other reagents were purchased from Aldrich
and were used as received. Bicinchoninic acid assay kit, Triton X-100 and Tween-20 were
from Thermo Scientific (Rockford, IL). Purified recombinant human LAL (phLAL) was a
gift of Drs. Greg Grabowski and Hong Du (Cincinnati Children's Hospital Medical Center,
1
13
OH) and was prepared as described.25 H and C spectra were obtained using a Varian
INOVA instrument operating at 600 MHz and 150 MHz, respectively. All spectral data are
1
reported in ppm (δ) relative to the residual solvent peaks present. For H NMR, the ppm are
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Rosenbaum et al.
Page 8
13
relative to 7.27 (CDCl ) and 2.5 (DMSO-d ). For C NMR, the ppm are relative to 77.23
3
6
(CDCl ) and 39.43 (DMSO-d ). Coupling constants (J) are reported in Hz. High resolution
3
6
mass spectrometry (HRMS) was performed on a JEOL GCMate system. Flash
chromatography was performed using EMD silica gel (200–400 mesh). Infrared spectra
were recorded on a Perkin Elmer Paragon 1000 FT-IR Spectrometer. All tested compounds
were assessed to be ≥ 95% pure by analytical HPLC.
Typical procedure for the synthesis of 4-amino-3-chloro-1,2,5-thiadiazoles. 3-
Pyrrolidinyl-4-chloro-1,2,5-thiadiazole (2)
3,4-Dichloro-1,2,5-thiadiazole (0.61 mL, 6.45 mmol) was added dropwise to pyrrolidine
(2.2 mL, 25.8 mmol) at 22 °C. The solution immediately began to warm and turn orange in
color. After 5 min, TLC (DCM) showed complete consumption of the starting thiadiazole.
The reaction mixture was poured into ice water and acidified to pH 2.0 by addition of
concentrated aq HCl. The mixture was extracted with DCM (2 × 10 mL) and the combined
organic extracts were dried with a SO , filtered, and evaporated. The product was purified
2
4
by column chromatography (DCM as eluent) to yield 1.07 g (87%) of the desired
1
product. H NMR (600 MHz, CDCl ) δ 3.72 (t, J = 7.2 Hz, 4H), 1.98 (t, J = 7.2 Hz,
3
13
4H); C NMR (150 MHz, CDCl ) δ 156.4, 131.2, 49.9, 25.9; HRMS (ESI) calcd for
3
+
−1
C H ClN S [M] 189.0127, found 189.0122; IR (neat): 2962, 2899, 2872, 1502 cm .
6
8
3
Typical procedure for the synthesis of 3-hydroxy-4-amino-1,2,5-thiadiazoles. 3-Hydroxy-4-
pyrrolidinyl-1,2,5-thiadiazole (6)
2 (600 mg, 3.16 mmol) was suspended in a H O/DMSO mixture (4:1, 5 mL) containing
2
KOH (710 mg, 12.65 mmol). The mixture was stirred under reflux for 4 h. After this time,
TLC (DCM) showed all starting thiadiazole to be consumed. The reaction was cooled to 0
°C. The solution was acidified to pH 2.0 by addition of concentrated HCl. A white
precipitate formed, which was filtered and washed with water. After drying, the product was
1
recrystallized from MeOH to yield 514 mg (95%) of a white solid. mp > 200 °C. H NMR
13
(600 MHz, DMSO-d ) δ 12.4 (bs, 1H), 3.54 (t, J = 7.2 Hz, 4H), 1.84 (t, J = 7.2 Hz, 4H);
C
6
NMR (150 MHz, DMSO-d ) δ 152.4, 148.7, 48.3, 24.8; HRMS (ESI) calcd for C H N OS
6
6 9 3
+
−1
[M + Na] 194.0359, found 194.0342; IR (KBr): 3227, 2961, 2859, 1501 cm .
Typical procedure for the synthesis of 3-carbamoyl-4-amino-1,2,5-thiadiazoles. 4-
(Pyrrolidin-1-yl)-1,2,5-thiadiazol-3-yl piperidine-1-carboxylate (10)
t
6 (70 mg, 0.406 mmol) was suspended in THF (1 mL) at 22 °C. KO Bu (46 mg, 0.406
mmol) was added as a solid, and the mixture was stirred for 10 min. Piperidinecarbonyl
chloride (0.04 mL, 0.339 mmol) was then added via syringe. The reaction was stirred for 12
h, during which time it became cloudy and turbid. Water (10 mL) was added and the
mixture was extracted with DCM (2 × 10 mL). The combined organic extracts were dried
with Na SO , filtered, and concentrated. The residue was dissolved in MeOH (1 mL) and
2
4
cooled to 0 °C. Water was added, and scratching with a spatula induced a white solid to
form. The solid was filtered, washed with water, and dried to yield 69 mg (72%) of the title
1
compound. mp = 57–60 °C. H NMR (600 MHz, CDCl ) δ 3.60-3.56 (m, 6H), 3.53-3.51 (m,
3
13
2H), 1.95 (t, J = 6.6 Hz, 4H), 1.68-1.60 (m, 6H); C NMR (150 MHz, CDCl ) δ 151.9,
3
151.5, 144.4, 48.5, 46.0, 45.7, 26.2, 25.7, 25.6, 24.3; HRMS (ESI) calcd for C H N O S
12 18
4 2
+
−1
[M + Na] 305.1043, found 305.1039; IR (film): cm .
Typical procedure for the reaction between 4-amino-3-chloro-1,2,5-thiadiazoles and
enolates. tert-Butyl 2-(4-morpholino-1,2,5-thiadiazol-3-yl)acetate (18)
Monochloride 4 (200 mg, 0.97 mmol) was charged into a flame-dried flask and evacuated
for 10 min. The flask was brought into a glovebox and LiHMDS (374 mg, 2.24 mmol) was
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Rosenbaum et al.
Page 9
added. The flask was capped, removed from the glovebox, and placed under argon. THF (3
mL) was added via syringe, and the solution immediately darkened in color. tert-Butyl
acetate (0.14 mL, 1.07 mmol) was then added via a syringe. The mixture was heated to 60
°C and monitored by TLC (DCM as eluent). Upon completion, the mixture was cooled to 22
°C and quenched with 1.0 N HCl. The mixture was extracted with Et O and the combined
2
organic extracts were dried with MgSO , filtered, and concentrated. The crude product was
4
purified by column chromatography (2% EtOAc in DCM) to furnish 222 mg (80%) of the
1
title compound as an oil. H NMR (600 MHz, CDCl ) δ 3.82 (m, 4H), 3.78 (s, 2H), 3.26 (m,
3
13
4H), 1.44 (s, 9H); C NMR (150 MHz, CDCl ) δ 168.1, 163.1, 148.3, 82.2, 66.6, 50.4,
37.9, 28.1; HRMS (ESI) calcd for C H N O S [M + Na] 308.1039, found 308.1002; IR
(neat): 2975, 2928, 2853, 1729, 1501, 1440 cm .
3
+
12 19
3 3
−1
1-Morpholino-2-(4-morpholino-1,2,5-thiadiazol-3-yl)ethanone (26)
18 (100 mg, 0.35 mmol) was dissolved in a 1:1 solution of DCM:TFA (2 mL) at 22 °C. The
solution was stirred for 3 h. After this time, TLC analysis (2% EtOAc in DCM) showed
complete consumption of the ester. The solution was evaporated to dryness, and the residue
was redissolved in DCM (2 mL). The flask was cooled to 0 °C and morpholine (37 mg, 0.42
mmol) was added via syringe, followed by EDC (202 mg, 1.05 mmol). The reaction mixture
was stirred for 12 h and gradually warmed to 22 °C. After this time, TLC analysis
(hexane:EtOAc 2:1) showed complete consumption of the starting material and a new more
polar spot. The mixture was diluted with DCM (10 mL) and washed with 1.0 N HCl (2 × 10
mL). The organic layer was dried with Na SO , filtered, and concentrated to yield a yellow
2
4
1
solid (82 mg, 78%). mp = 66–68 °C. H NMR (600 MHz, CDCl ) δ 3.88 (s, 2H), 3.84-3.82
(m, 4H), 3.68-3.65 (m, 4H), 3.63-3.62 (m, 2H), 3.53-3.56 (m, 2H), 3.33-3.32 (m, 4H);
3
13
C
NMR (150 MHz, CDCl ) δ 166.8, 163.5, 148.9, 66.9, 66.79, 66.77, 50.7, 47.0, 42.6, 36.2;
3
+
HRMS (ESI) calculated for C H N O S [M + Na] 321.0992, found 321.1001; IR (film):
2958, 2919, 2850, 1640, 1501, 1437, 1112 cm .
12 18
4 3
−1
Cell lines
Human fibroblast cell lines GM03123 (NPC1) and GM05659 (apparently normal) were
obtained from the Coriell Institute (Camden, NJ). Fibroblasts were grown in Minimal
Essential Medium (MEM) with 2.2 g/L sodium bicarbonate, supplemented with 10 % (v/v)
FBS (growth medium).
LAL activity assay
LAL activity was determined mainly as described previously.12 For IC (compound
50
concentration required for 50 % inhibition of activity) determinations, three-fold serial
dilutions of the compounds were prepared from DMSO stocks and diluted in the assay
buffer (200 mM sodium acetate, pH 5.5, 0.02 % (w/v) Tween-20). Purified enzyme (phLAL,
1 mU/ml final concentration, 105 U/mg, 1 U of enzyme releases 1.0 μmol of 4-
methylumbelliferone per minute at pH 5.5 at 37 °C) was then added. The enzyme/compound
mixture was incubated at 37 °C for 30 min. The reaction was started by addition of 125 μM
(final) 4-methylumbelliferyloleate (4MUO) dissolved in 4 % (w/v) Triton X-100. Reactions
were incubated at 37 °C, and fluorescence (355 nm excitation/450 nm emission) was
monitored using a SpectraMax M2 fluorometer (MDS Inc., Toronto, Canada). For the IC
50
determinations, the reaction time was 1 h. IC 's for selected compounds were determined
50
by fitting (MATLAB, Non-linear least squares Trust-Region algorithm) dose-response
curves to the rectangular hyperbola y=m/(x+b)+c, solved for y = 50, where y = normalized
enzymatic activity (%), x = compound concentration (nM), and m, b, c are coefficients.
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Page 10
Decarbamoylation rates (k ) were determined as follows: phLAL was incubated with 10 μM
3
compound for 30 min, diluted 250× to 40 nM with substrate (125 μM). The reaction was
monitored at 10 min intervals for 2 h. The apparent decarbamoylation rates were calculated
in MATLAB via linear regression of the plots of the equation as described previously16:
ln(Fraction Inhibited) = −k*t, Fraction Inhibited = (Activity
-Activity
)/
DMSO
Compound
Activity
for each time point.
DMSO
phLAL carbamoylation was assessed by pre-incubating phLAL with the various compounds
at 833 nM for indicated times at 37 °C. The reaction was started by addition of 125 μM
(final) 4-methylumbelliferyloleate (4MUO) dissolved in 4 % (w/v) Triton X-100 and
monitored for 30 min at 37 °C. The final compound concentration in the assay was 500 nM.
LAL activity in lysed cells was determined mostly as described previously.12
Human fibroblasts were grown in 384-well assay plates (Corning), incubated with
compounds for 3–4 days at indicated concentrations. At the end of the incubation, the cells
were washed 3× with PBS, then washed once with 150 mM sodium acetate pH 5.5, and then
lysed with 1 % (w/v) Triton X-100 for 30 min at 37 °C. The reaction was started by the
addition of 1 mM (final) 4MUO dissolved in 4 % Triton X-100. The reaction was monitored
as described above. Enzymatic activity was normalized to protein concentration in the
reaction mixture as determined by the bicinchoninic acid assay. No significant variation in
protein concentration between the different conditions was observed.
Automated fluorescence microscopy and image analysis
Filipin and LipidTOX green staining and automated image analysis (LSO filipin assay) were
MICRO
done as described.12 Images were acquired using an ImageXpress
imaging system
from Molecular Devices Devices (MDS Analytical Technologies, Downington, PA, USA)
equipped with a 300 W Xenon-arc lamp. A CoolSnapHQ camera (1,392 × 1,040 pixels)
from Roper Scientific (Tucson, AZ, USA) was used to acquire images. Filipin images were
acquired using 377/50 nm excitation and 447/60 nm emission filters with a 415 nm dichroic
filter using a 10× Plan Fluor 0.3 numerical aperture (NA) objective from Nikon (Melville,
NY, U.S.A). LipidTOX green images of neutral lipid accumulation were acquired using
482/35 nm excitation and 536/40 nm emission band-pass filters with a 513 nm dichroic filter
using a 20× Plan Fluor 0.5 NA objective from Nikon (Melville, NY, U.S.A). Images were
acquired at either four (10×) or nine (20×) sites per well using 2 × 2 pixel binning. Each site
was individually focused using a high-speed laser autofocus comprised of a 690 nm diode
laser and a dedicated 8-bit CMOS camera. 696 × 520 pixel images were acquired at 12
intensity bits per pixel.
Images were background corrected as described and either the filipin or the LipidTOX green
LSO ratio was quantified as integrated thresholded fluorescence power of filipin or
LipidTOX green fluorescence within the LSOs, normalized to the total cell area as defined
by a low threshold using filipin fluorescence.10
Software
MetaExpress and MetaMorph were from Molecular Devices (MDS Analytical
Technologies, Downington, PA, USA). MATLAB was from The MathWorks (Natick, MA).
Compound structures were generated using ChemDraw (CambridgeSoft, Cambridge, MA).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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Rosenbaum et al.
Page 11
Acknowledgments
The authors thank the Ara Parseghian Medical Research Foundation and NIH grant R37-DK27083 (to FRM) for
financial support of this research. We thank Dr. Peter Lobel (Rutgers University) for helpful discussions and Harold
Ralph (Weill Cornell Cell Screening Core Facility) for help with HTS image acquisition and analysis. We also
thank Drs. Gregory Grabowski and Hong Du (Cincinnati Children's Hospital Medical Center) for a gift of phLAL.
References
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Scheme 1.
a
Reagents and conditions: (a) 2° amine (4 equiv), 22 °C or Δ; (b) KOH (4 equiv),
t
DMSO:H O, reflux; (c) KO Bu, aminocarbonyl chloride, THF, 22 °C.
2
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Scheme 2.
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Page 15
Scheme 3.
a
Reagents and conditions: (a) 1.0 equiv LiHMDS, THF, 22 °C, 3 min; (b) 1.3 equiv
t
t
LiHMDS, BuOAc, THF, 60 °C, 77%; (c) 2.3 equiv LiHMDS, 1.1 equiv BuOAc, THF, 60
°C, 80%.
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Scheme 4.
Kinetic outline of transient LAL modification by 3,4-disubstituted thiadiazole carbamates.
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Page 17
Figure 1.
An example of LAL inhibitor previously observed to reduce lysosomal cholesterol levels in
NPC-deficient cells.
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Page 18
Figure 2. Lineweaver-Burk analysis of LAL inhibition
Compound 13 was pre-incubated for 30 min at 37 °C with phLAL, the reaction was started
by addition of 4MUO at various concentrations and monitored for 45 min. Compound
concentration indicated refers to the concentration in the final reaction mixture. Linear
2
regression fits to the data were computed in MATLAB. R >0.95 for all fits. Data are from
two independent experiments (6<n<8 for treated samples, where n is total number of wells
per condition used for quantification).
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Page 19
Figure 3.
Time-dependence of LAL inhibition by thiadiazole carbamates. phLAL was pre-incubated
with the various compounds at 833 nM for the indicated times at 37 °C. The reaction was
started by addition of 125 μM 4MUO and monitored for 30 min at 37 °C. The final
compound concentration in the assay was 500 nM. Data represent averages ± SEM of 2
independent experiments normalized to control (DMSO) average value for each experiment
(n=12, where n is total number of wells per condition used for quantification).
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Rosenbaum et al.
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Figure 4.
Compound efficacy in cells. Enzymatic assay for inhibition of LAL in cell lysates of cells
treated with compound continuously for ~4 days (A) or treated for ~3 days and allowed to
recover in growth media for ~1 additional day (B). Data represent averages ± SEM of 3
independent experiments normalized to control (DMSO) average value for each experiment
(n=12, where n is total number of wells per condition used for quantification).
Quantification of thiadiazole effects after ~3 days of treatment on cholesterol accumulation
as measured by filipin LSO assay in NPC1 mutant cells (C) or neutral lipid accumulation in
apparently normal human fibroblasts as measured by quantification of LipidTOX green
staining (D). Data are presented as percentage of the average value of the DMSO controls
(C) or normalized to the 10 μM 12 average value (D) in each experiment. Error bars, where
visible, are SEM. (n=8 for treated samples, where n is total number of wells per condition
used for quantification).
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Table 1
IC values and decarbamoylation rates for thiadiazoles produced via Scheme 1.
50
a
IC50 (nM)
Compound
S.E.M. decarbamoylation rate (min−1 b
)
S.E.M.
0.0001
0.0002
0.0001
0.0002
0.0001
0.0002
0.0001
0.0001
10
11
12
13
14
15
16
17
496
34
62
50
21
53
69
106
47
0.0007
473
0.0037
0.0010
0.0030
0.0016
0.0043
0.0013
0.0042
152
68
424
492
300
189
a
When appropriate, apparent IC 's for selected compounds against purified human LAL were determined by fitting (MATLAB, non-linear least
50
squares trust-region algorithm) dose-response curves (obtained with the 4MUO assay) to the rectangular hyperbola y=m/(x+b)+c, for y=50, where
2
y = normalized enzymatic activity [%] and x = compound concentration [nM], and m, b, c are coefficients. All fits had R > 0.95. Data represent
averages ± SEM of 3 independent experiments.
b
phLAL was incubated with 10 μM compound for 30 min, diluted 250× to 40 nM with substrate (0.125 mM). The reaction was monitored at 10
−1
min intervals for 2 hours. The apparent decarbamoylation rates [min ] were calculated in MATLAB via linear regression of the plots of the
equation described previously:16 ln(Fraction Inhibited) = −k*t, Fraction Inhibited = (Activity
-Activity )/Activity for
Compound DMSO
DMSO
2
each time point. Data represent averages ± SEM of 4 independent experiments. R >0.96 for all fits.
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Table 2
The synthesis of C(3) methylene analogues.
Compound
18
R
a
Yield
80%
69%
65%
19
20
21
22
90%
77%
b
83%
71%
82%
23
b
24
25
Footnotes:
a
Refers to isolated, purified yields.
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Page 23
b
Used LDA (1.2 equiv) to preform the enolate.
J Med Chem. Author manuscript; available in PMC 2011 July 22.