Design, synthesis, and pharmacological evaluation of bis-2-(5- phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) analogs as glutaminase inhibitors

Article abstract of DOI:10.1021/jm301191p

Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) is a potent and selective allosteric inhibitor of kidney-type glutaminase (GLS) that has served as a molecular probe to determine the therapeutic potential of GLS inhibition. In an attempt to identify more potent GLS inhibitors with improved drug-like molecular properties, a series of BPTES analogs were synthesized and evaluated. Our structure-activity relationship (SAR) studies revealed that some truncated analogs retained the potency of BPTES, presenting an opportunity to improve its aqueous solubility. One of the analogs, N-(5-{2-[2-(5-amino-[1,3,4] thiadiazol-2-yl)-ethylsulfanyl]-ethyl}-[1,3,4]thiadiazol-2-yl) -2-phenyl-acetamide 6, exhibited similar potency and better solubility relative to BPTES and attenuated the growth of P493 human lymphoma B cells in vitro as well as in a mouse xenograft model.

Full text of DOI:10.1021/jm301191p

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Article
Design, synthesis, and pharmacological evaluation
of bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl
sulfide (BPTES) analogs as glutaminase inhibitors
Krupa Shukla, Dana V. Ferraris, Ajit G Thomas, Marigo Stathis, Bridget Duvall, Greg Delahanty, Jesse Alt,
Rana Rais, Camilo Rojas, Ping Gao, Yan Xiang, Chi Van Dang, Barbara S. Slusher, and Takashi Tsukamoto
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301191p • Publication Date (Web): 14 Nov 2012
Downloaded from http://pubs.acs.org on November 25, 2012
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Design, synthesis, and pharmacological evaluation
of bisꢀ2ꢀ(5ꢀphenylacetamidoꢀ1,2,4ꢀthiadiazolꢀ2ꢀ
yl)ethyl sulfide (BPTES) analogs as glutaminase
inhibitors
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Krupa Shukla, Dana V. Ferraris, Ajit G. Thomas, Marigo Stathis, Bridget Duvall, Greg
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Delahanty, Jesse Alt, Rana Rais, Camilo Rojas, Ping Gao, Yan Xiang, Chi V. Dang,
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*,†,‡
Barbara S. Slusher, and Takashi Tsukamoto
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‡
Department of Neurology and Brain Science Institute, Johns Hopkins University, Baltimore,
MD 21205, USA
School of Life science, University of Science and Technology of China, Hefei 230027, China
║
Abramson Family Cancer Research Institute, Abramson Cancer Center of the University of
Pennsylvania, Philadelphia, PA 19104, USA
CORRESPONDING AUTHOR *Phone: (410) 614ꢀ0982. Fax: (410) 614ꢀ0659. Eꢀmail:
ttsukamoto@jhmi.edu.
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ABSTRACT: Bisꢀ2ꢀ(5ꢀphenylacetamidoꢀ1,2,4ꢀthiadiazolꢀ2ꢀyl)ethyl sulfide (BPTES) is a potent
and selective allosteric inhibitor of kidneyꢀtype glutaminase (GLS) that has served as a
molecular probe to determine the therapeutic potential of GLS inhibition. In an attempt to
identify more potent GLS inhibitors with improved drugꢀlike molecular properties, a series of
BPTES analogs were synthesized and evaluated. Our structureꢀactivity relationship (SAR)
studies revealed that some truncated analogs retained the potency of BPTES, presenting an
opportunity to improve its aqueous solubility. One of the analogs, Nꢀ(5ꢀ{2ꢀ[2ꢀ(5ꢀaminoꢀ
[1,3,4]thiadiazolꢀ2ꢀyl)ꢀethylsulfanyl]ꢀethyl}ꢀ[1,3,4]thiadiazolꢀ2ꢀyl)ꢀ2ꢀphenylꢀacetamide,
exhibited similar potency and better solubility relative to BPTES and attenuated the growth of
P493 human lymphoma B cells in vitro as well as in a mouse xenograft model.
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INTRODUCTION
Glutaminase catalyzes the hydrolysis of glutamine into glutamate and ammonia. Mammalian
tissues express two isoforms of glutaminase that are derived from distinct but structurally related
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genes. Kidneyꢀtype glutaminase (GLS) is widely distributed throughout extraꢀhepatic tissues
whereas liverꢀtype glutaminase (GLS2) is found primarily in adult liver. GLS plays a critical role
in glutaminolysis as an important energy source for many proliferating cells, especially rapidly
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growing malignant cells.
Recent studies have shown that Myc, an oncogenic transcription
factor, increases glutamine metabolism by stimulating the expression of glutaminase directly or
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via suppression of miR23a and miR23b. It is conceivable that, under hypoxic conditions,
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glutaminolysis provides the dominant means for cancer cell metabolism. Indeed, in both P493
human B lymphoma cells and PC3 prostate cancer cells, the use of GLS siRNA diminished
glutaminase levels and resulted in a remarkable decrease in cell proliferation. The
antiproliferative effects could be recapitulated by removal of glutamine from the cell culture
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medium. These findings indicate that small molecule GLS inhibitors might exert
antiproliferative effects against Mycꢀdependent and other cancers.
In the nervous system, GLS is responsible for the production of intracellular glutamate, a key
excitatory neurotransmitter, as a crucial part of the glutamineꢀglutamate cycle. However,
accumulating evidence suggests glutamate formed by upregulated GLS in activated macrophages
and microglia is playing a key pathogenic role in inflammatory neurological disorders such as
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HIV1ꢀassociated dementia and multiple sclerosis. Therefore, small molecule GLS inhibitors
may offer therapeutic potential in these devastating neurodegenerative diseases by reducing
excess extracellular glutamate.
Despite the therapeutic potential of GLS inhibition, little effort has been made to explore the
therapeutic utility of GLS inhibition for two key reasons. First, GLS plays a critical role in
cellular metabolism, thus GLS inhibition could have major undesired physiological
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consequences. Indeed, recent studies showed that GLS knockouts die shortly after birth.
However, mice heterozygous for the GLS (GLS hets) are fully viable despite a mild yet
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significant reduction in glutamate levels in some tissues. These findings suggest that GLS
inhibition may provide a viable therapeutic option particularly for the treatment of cancer, where
the clinical benefit outweighs the risk associated with a narrow therapeutic index. Second, little
progress has been made in identifying potent and selective inhibitors of GLS. Although a
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conventional GLS inhibitor 1, known as 6ꢀdiazoꢀ5ꢀoxoꢀLꢀnorleucine (DON), has been
extensively utilized as a tool for studying physiological roles of GLS, its inherent chemical
reactivity coupled with lack of selectivity and poor potency has hampered its use in establishing
the therapeutic benefit of selective GLS inhibition.
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The renewed interest in GLS as a therapeutic target in recent years has prompted efforts to
identify novel glutaminase inhibitors. For instance, dibenzophenanthridines represented by
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compound 2 are being explored as a new class of GLS inhibitors for the treatment of cancer.
Compound 3, known as bisꢀ2ꢀ[5ꢀ(phenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl]ethyl sulfide or
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BPTES, represents another class of GLS inhibitors structurally distinct from 1 (Figure 1).
Compound 3 offers some fundamental advantages over compounds 1 and 2. Unlike 1, compound
does not contain any reactive chemical group that might form a covalent bond and is unlikely
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to cause toxicity by irreversibly forming covalent adducts with endogenous proteins. Moreover,
compound 3 bears no structural similarity to either glutamine or glutamate. This should minimize
toxicological risk due to its interaction with other enzymes, transporters, or receptors that
recognize glutamine or glutamate as substrates. For example, unlike 1, compound 3 selectively
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inhibits GLS over GLS2 and γꢀglutamyl transpeptidase. A substrate concentrationꢀdependent
kinetic study revealed that 3 inhibits GLS in a uncompetitive manner by facilitating the
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formation of an inactive tetramer.
In addition compound 3 is capable of inhibiting the
phosphateꢀactivated GLS while compound 2 shows little ability to inhibit GLS in the presence of
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phosphate. Recently, three independent groups solved crystal structures of GLS.
Some of
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these structures were determined as complexes with compound 3,
providing compelling
evidence for the previously proposed inactive tetramer formation by compound 3. The new
structural knowledge offers distinct advantages of compound 3 over other classes of GLS
inhibitors as a template to conduct more rational structural optimizations.
Because of its unique biochemical characteristics, compound 3 has been increasingly utilized
to study the physiological role of GLS as well as the potential therapeutic effects of GLS
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inhibition.
Compound 3, however, is highly hydrophobic with an aqueous solubility of less
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than 1 ꢀg/mL. The poor solubility hinders its utility as a pharmacological probe and prompted us
to explore whether structural modifications can be made to compound 3 without sacrificing its
GLS inhibitory potency.
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Herein is described systematic structureꢀactivity relationship (SAR) analysis of analogs
derived from 3 as a part of our ongoing efforts to identify drugꢀlike GLS inhibitors. A series of
truncated analogs were examined in an attempt to reduce the hydrophobicity and identify the
minimal structural requirements for GLS binding. One of the truncated analogs 6 was also tested
for its ability to attenuate lymphoma cell (P493) proliferation in vitro and tumor xenograft
growth in vivo.
CHEMISTRY
Two phases of SAR studies were carried out for analogs of compound 3. First, various
truncated analogs were explored to identify the pharmacophore required for GLS binding.
Second, one of the truncated analogs was used as a template for further structural optimization in
an attempt to improve the potency against GLS.
Scheme 1 illustrates the synthesis of 3 as well as its truncated analogs 5, 6, and 7. Reaction of
thiodipropionic acid 4 with thiosemicarbazide in the presence of phosphorous oxychloride gave
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bisꢀaminothiadiazole 5.
Compound 3 was obtained by coupling 5 with 2.0 equivalents of
phenylacetyl chloride. On the other hand, monoacylated compound 6 was obtained by reacting 5
with only 1.0 equivalent of phenylacetyl chloride. Sulfoxide 7 was prepared from sulfide 6 using
hydrogen peroxide.
Truncated analogs lacking a sulfide group in the linker region (compounds 11a and 11b) were
prepared from acid chlorides 8a and 8b, respectively (Scheme 2). Reaction of 8 with
thiosemicarbazide in the presence of phosphorous oxychloride followed by aqueous workꢀup
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provided aminothiadiazoleꢀcontaining carboxylic acids 9. The aminothiadiazole group of 9 was
acylated with phenylacetyl chloride to give 10. Subsequent reaction with thiosemicarbazide in
the presence of phosphorous oxychloride afforded the desired products 11a and 11b.
Compound 9b also served as an intermediate for analogs of 11b where its phenylacetyl group
is replaced with other acyl groups (Scheme 2). Compound 9b was first converted to the
corresponding methyl ester 12. Subsequent reaction with acyl chlorides followed by hydrolysis
gave 14a–f. Reaction of 14a–f with thiosemicarbazide in the presence of phosphorous
oxychloride afforded the desired products 15a–f.
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Schemes 3 illustrates the synthesis of compounds 20, where the internal thiadiazole ring of 11b
is replaced by a thiazole ring. Methyl adipoyl chloride 8b was treated with diazomethane to
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afford an intermediate αꢀchloroketone 16, which was reacted with thiourea to form 17. The
aminothiazole 17 was converted into the desired compound 20 by acetylation, hydrolysis and
cyclization. Compound 27, another thiazoleꢀcontaining analog, was synthesized from acid
chloride 21 as shown in Scheme 4. Rosenmund reduction of 21 gave aldehyde 22, which was
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brominated to afford αꢀbromoaldehyde 23. The bromide 23 was cyclized with thiourea to
produce the desired aminothiazole 24, which was converted to analog 27 in a manner similar to
the preparation of 20 from 17.
As shown in Scheme 5, compounds 29a–m, in which the terminal thiadiazole ring of 11b is
replaced by amide group, were prepared by coupling 10b with a variety of amines 28a–m using
HATU as a coupling reagent.
RESULTS AND DISCUSSION
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All new synthetic compounds were tested for their ability to inhibit GLS using Lꢀ[ H]ꢀ
glutamine as substrate and human kidneyꢀtype glutaminase (hKGA124ꢀ669). Table 1 summarizes
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the GLS inhibitory potency of the truncated analogs of 3. The removal of both phenylacetyl
groups resulted in a significant loss of potency (compound 5). A similar trend (50ꢀfold increase
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in IC value from 3 to 5) has been previously reported. Removal of one phenylacetyl group
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from 3 (compound 6), however, did not result in the significant change in the GLS inhibitory
potency. Potent inhibition of GLS by an asymmetric compound such as 6 is rather surprising
because the crystal structures of GLS in complex with symmetric compound 3 revealed that each
identical half of compound 3 interacts with each GLS monomer in a highly symmetrical
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fashion.
While the possibility of a distinct binding site and/or mode for compound 6 cannot
be completely ruled out, prior crystallographic studies have demonstrated that both compounds 3
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Figure 2A) and 5 (Figure 2B) bind to the same allosteric site of GLS. Given the structure of 6
being an intermediate between compounds 3 and 5, it is conceivable that compound 6 binds to
the same allosteric site and preserves some of the key interactions with GLS that were
indentified in the crystallographic studies of compounds 3 and 5. Furthermore, we conducted
inhibitory kinetic studies of compound 6 at various concentrations of glutamine to determine its
precise mode of GLS inhibition. As shown in Figure 3, a double reciprocal plot of glutaminase
activity versus the glutamine concentrations produced parallel lines for different concentrations
of compound 6, indicative of uncompetitive inhibition with a K value of 2.0 ꢁM. This is
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consistent with the mode of inhibition reported for compound 3 and provides further supporting
evidence that compounds 3 and 6 shares the same allosteric binding site of GLS. It remains to be
explored how asymmetric compounds such as 6 can bind to the highly symmetric GLS dimer
interface region and lock the enzyme into an inactive conformation. A relevant precedent can be
found in HIVꢀ1 protease which forms a symmetrical dimer and yet accommodates both
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symmetric and asymmetric inhibitors. Nevertheless, this SAR trend presented a promising
opportunity to identify GLS inhibitors of lower molecular weight and hydrophobicity.
Subsequently, we conducted further structural optimization of compound 6. In an attempt to
improve aqueous solubility, sulfoxide analog 7 was prepared, though it turned out to be a weak
inhibitor of GLS with an IC value of 61 ꢂM. In the next set of SAR studies, we tested analogs
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of 6, in which the middle linker was shortened by removing sulfide and one methylene unit
compound 11a) or sulfide only (compound 11b). While significant loss of GLS inhibitory
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potency was observed with 11a, compound 11b exhibited potency similar to 6, indicating that
the middle linker can be shortened to some degree without compromising the affinity to GLS.
This is consistent with the crystal structure of KGA complexed with 5,5'ꢀbutaneꢀ1,4ꢀ
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diylbis(1,3,4ꢀthiadiazolꢀ 2ꢀamine), another 4ꢀcarbon linker containing GLS inhibitor (3VP4).
The linker was able to adopt stretched conformation so that the two thiadiazole rings can be
positioned in a manner similar to those of compound 3.
Compounds 15a–f represents a variety of analogs in which the phenylacetyl group of 11b was
replaced by various acyl groups. With the exception of compound 15a, modifications at this site
were generally well tolerated with IC values ranging from 2.6 to 12 ꢂM. Only modest loss of
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potency in compound 15c bearing a cyclohexylacetyl group indicates that the terminal aromatic
ring is not an essential component for GLS inhibition. In fact, crystal structures indicate that the
phenyl groups of compound 3 do not form piꢀstacking interactions with any aromatic residues of
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GLS (Figure 2A). The flat SAR trend within the various amide analogs may be attributed to the
lack of specific interaction between the terminal acyl group and the allosteric site.
In compounds 20 and 27, the internal thiadiazole group of 11b was replaced by a 1,3ꢀthiazole
ring. Neither compounds inhibited GLS up to 100 ꢂM. The complete loss of potency seen in
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compound 27 particularly highlights the crucial role played by N of the thiadiazole ring most
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likely through its interaction with Phe322 and/or Tyr394 residue(s). Indeed, mutant forms of
GLS (F322S, Y394I, or Y394L) exhibited similar enzymatic activity compared to the wildꢀtype
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form but showed significantly reduced sensitivity to 3,
illuminating an important role played
by these residues in the allosteric binding site of GLS.
Subsequent to the identification of 11b as a potent GLS inhibitor, we explored the structural
modification of its terminal thiadiazole ring. In analogs 29a–m, the terminal thiadiazole of 11b
was replaced by a variety of amide groups. As shown in Table 2, some of these compounds
exhibited potent GLS inhibitory activity. Most notable inhibitors were amides 29a and 29c–f
which contain a pyrrolidine or piperidine moiety. These compounds inhibited GLS with IC50
values of 10 ꢀM or below. It appears the cyclic amino group is preferred for GLS inhibitory
activity as analogs 29g and 29h lacking a ring system showed significantly lower potency.
Among other amides synthesized, only tertꢀbutyl amide derivative 29i exhibited reasonably
potent GLS inhibition. Tertiary amide 29b was completely devoid of GLS inhibitory activity
while another tertiary amide 29j was equally potent as its close analog 29i. Overall, these
findings demonstrate that the terminal thiadiazole can be replaced without significant loss of
potency. While the mode that these amide groups interact with the allosteric binding site of GLS
remains unclear, it is conceivable that the extra nitrogen atom of 29a and 29c–f participates in
hydrogen bonding with some of the key residues (Phe322, Tyr394, Leu323) identified in
complexes of GLS with compound 3 (Figure 2A). Since this series of compounds can be
synthesized from the common intermediate 10b in one step, SAR studies can be extended to a
variety of amines to provide further insights into the molecular features required for the high
affinity to GLS.
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To examine the effect of the structural modifications on the physicochemical properties, we
assessed the aqueous solubility of GLS inhibitors 6, 11b, and 29a relative to that of compound 3.
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Using the LC/MS based shakeꢀflask method, aqueous solubility of 3, 6, 11b and 29a was
determined to be 0.144, 13, 3.4 and 683 ꢀg/mL, respectively. The improved solubility over
compound 3 makes the new GLS inhibitors not only more valuable pharmacological tools but
also more tractable leads for further structural optimization.
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Subsequent pharmacological characterization was conducted with one of the truncated analogs,
compound 6. At concentrations up to 100 ꢂM, compound 6 showed no inhibition of mouse liverꢀ
type glutaminase (mouse GLS2), whose sequence is highly homologous to that of a human
24
GLS2. Although speculative, it is conceivable that the highly specific inhibition of GLS by
compound 6 can be attributed to its ability to retain the critical interaction with Phe322, one of
the residues unique to GLS allosteric loop. In vitro metabolic stability studies using mouse liver
microsomes showed the loss of compound 6 over time (42 and 16% remaining after 30 and 60
min incubation, respectively) in the presence of NADPH. LC/MS analysis identified compound 7
as one of the major metabolites which formed through the oxidation of the sulfide group in the
linker region of compound 6. Consistent with the metabolic site of 6, nonꢀsulfide GLS inhibitors
11b and 29a were found to be completely stable against microsomes in the presence of NADPH.
Since compound 6 possesses a primary amino group at its terminal thiadiazole ring,
glucuronidation might also play a role in the liver metabolism of this compound. Fortification of
the microsome incubation with UDGPA, however, resulted in negligible loss of the parent
compound.
Antitumor effects of compound 6 was assessed using the human P493 B cells, in which Myc is
2
5
overꢀexpressed in the absence of tetracycline. As shown in Figure 4A, compound 6 attenuated
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the growth of P493 cells in vitro in a doseꢀdependent manner with an IC value near 20 ꢂM.
5
0
Previous studies have shown that similar antiproliferative effects can be achieved by GLS
4
4
18
siRNA, glutamine deprivation, or treatment with compound 3. In the same assay, compound
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2
9a inhibited the growth with IC50 value near 40 ꢁM. An attempt to assess antiproliferative
activity of compound 11b was unsuccessful due to its poor aqueous solubility.
Compound 6 was also tested in an animal model in which compound 3 was previously
18
reported to exhibit antiproliferative efficacy. Given the instability in liver microsomes,
intraperitoneal injection was chosen as a route of administration to avoid the first pass
metabolism. As shown in Figure 4B, P493 tumor xenograftꢀbearing SCID mice treated with
compound 6 showed significantly diminished tumor growth compared to the vehicleꢀtreated
mice. The degree of efficacy achieved by compound 6 is comparable with that of compound 3
1
8
reported previously. As mentioned before, compound 6 can be metabolized in liver into the
inactive metabolite 7. Similar metabolism is expected for compound 3 since it also contains a
metabolically liable sulfide group. It would be interesting to test equally potent nonꢀsulfide GLS
inhibitors such as 11b and 29a to determine the effects of improved metabolic stability on the
degree of efficacy.
CONCLUSIONS
While kidneyꢀtype glutaminase (GLS) has recently gained increasing attention as a therapeutic
target for the treatment of cancer and neuroinflammatory diseases, little progress has been made,
to date, in efforts to identify new GLS inhibitors mechanistically distinct from the conventional
inhibitor 1. Allosteric GLS inhibitor 3 has presented an unprecedented opportunity to address the
needs to identify a new series of inhibitors selective to GLS over GLS2. Recently reported
crystal structures of GLS, particularly those in complex with compound 3, has provided new
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insights into the allosteric binding mode of compound 3 and its implication for the design of new
inhibitors. Incremental truncation of compound 3 led us to identify analogs equally potent as
compound 3 with improved solubility. While it is promising that the new GLS inhibitor 6 retains
antiꢀproliferative activity of compound 3 both in vitro and in vivo, further improvement,
particularly in potency, is necessary to translate GLS inhibitors into practical therapeutic
interventions. Although there was no apparent toxicity following the repeated intraperitoneal
injection of compound 6 in P493 tumor xenograftꢀbearing SCID mice, more extensive toxicity
studies are required to better define the therapeutic window of GLS inhibitors. Such
investigation, however, should await GLS inhibitors with better potency and ADME profile.
SAR and metabolic stability data provided herein coupled with the new structural insights gained
from the recently published GLS crystal structures should allow more refined structural
optimization to maximize the interaction with the allosteric binding site, leading to the discovery
of more desirable GLS inhibitors.
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EXPERIMENTAL SECTION
General. All solvents were reagent grade or HPLC grade. Unless otherwise noted, all
materials were obtained from commercial suppliers (Aldrich, Sigma, TCI, or Matrix) and used
without further purification. All reactions were performed under nitrogen. Preparative HPLC
purification was performed on an Agilent 1200 Series HPLC system equipped with a multiꢀ
wavelength detector using a Phenomenex Luna 5 ꢁm C18 (2) column (250 × 4.6 mm) with a
flow rate of 15 mL/min. The solvent system consisted of distilled water with 0.1% formic acid
(solvent A) and acetonitrile with 0.1% formic acid (solvent B). 40% B was used from 0 to 5 min
followed by a linear gradient of 40% to 85% B over 35 min. Melting points were obtained on a
1
13
MelꢀTemp apparatus and are uncorrected. H NMR spectra were recorded at 400 MHz. C NMR
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spectra were recorded at 100 MHz. Elemental analyses were obtained from Atlantic Microlabs,
Norcross, GA. The purity of test compounds was determined by elemental analysis (within
±
0.4% of the calculated value).
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12
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N,N'ꢀ(5,5'ꢀ(2,2'ꢀthiobis(ethaneꢀ2,1ꢀdiyl))bis(1,3,4ꢀthiadiazoleꢀ5,2ꢀdiyl))bis(2ꢀphenylacetamide)
1
1, 16
(3). The preparation of 3 from bisthiadiazole 5 has already been reported.
We used a similar
procedure to synthesize 3. To a solution of compound 5 (3.61 g, 12.5 mmol) in DMA (100 mL) was
added NEt (3.8 mL, 27.5 mmol) and phenylacetyl chloride (3.2 mL, 25 mmol). The reaction was
3
stirred overnight at room temperature. DMA was removed in vacuo and the resulting solid was
triturated with 10% NaHCO and boiling MeOH to obtain 3 as a white solid (5.8 g, 88% yield): mp
3
1
2
31–234 °C; H NMR (DMSOꢀd ) δ 2.92 (t, J = 7.2 Hz, 4H), 3.24 (t, J = 7.1 Hz, 4H), 3.79 (s, 4H),
6
13
7
1
1
.23–7.34 (m, 10H), 12.69 (s, 2H); C NMR (DMSOꢀd ) δ 29.4, 30.1, 41.6, 126.9, 128.5, 129.3,
6
34.7, 158.6, 162.3, 169.4. Anal. Calcd. for C H N O S ꢃ0.5H O: C, 54.01; H, 4.72; N, 15.75; S,
24 24
6
2
3
2
8.02. Found: C, 54.00; H, 4.54; N, 16.0; S, 18.41.
Bisꢀ[2ꢀ(5ꢀAminoꢀ[1,3,4]thiadiazolꢀ2ꢀyl)ꢀethylsulfide] (5). The preparation of compound 5 from
1
6
thiodipropionic acid 4 has already been reported. We used a similar procedure to synthesize 5.
Thiodipropionic acid 4 (10 g, 56 mmol) and thiosemicarbazide (10 g, 56 mmol) were dissolved in
POCl (30 mL) and heated to 90 °C for 3 h. The reaction was cooled and the viscous oil was poured
3
onto ~100 g ice. The filtrate was basified using solid NaOH until pH 10. The solid that precipitated
out of solution was filtered off and washed several times with H O. The solid was then resuspended
2
in boiling MeOH and filtered hot. This final filtration led to desired bisꢀaminothiadiazole 5 as a
1
white solid (12.7 g, 79% yield): mp 222 °C (dec); H NMR (DMSOꢀd ) δ 2.84 (t, J = 7.2 Hz, 4H),
6
13
3
.06 (t, J = 7.2 Hz, 4H), 7.04 (s, 4H); C NMR (DMSOꢀd ) δ 30.0, 30.3, 156.4, 168.5. Anal. Calcd.
6
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
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3
3
4
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5
5
5
5
5
6
for C H N S ꢃ0.1H Oꢃ0.02MeOH: C, 33.12; H, 4.26; N, 28.89; S, 33.07. Found: C, 33.49; H, 4.11;
8
12
6
3
2
N, 28.51; S, 32.84.
Nꢀ(5ꢀ{2ꢀ[2ꢀ(5ꢀAminoꢀ[1,3,4]thiadiazolꢀ2ꢀyl)ꢀethylsulfanyl]ꢀethyl}ꢀ[1,3,4]thiadiazolꢀ2ꢀyl)ꢀ2ꢀ
phenylꢀacetamide (6). Compound 5 (5.0 g, 17.3 mmol) was dissolved in hot DMA (~20–30 mL)
until in solution. The solution was cooled down and triethylamine (7.25 mL, 52 mmol) was added
followed by dropwise addition of phenyl acetyl chloride (2.6 g, 17.3 mmol). After stirring for 3 h,
the reaction was concentrated in vacuo to remove DMA and the residue was triturated with water
(~100 mL). The resulting solid was triturated with boiling THF (~300 mL). The insoluble solid was
filtered off (starting material) and the filtrate was concentrated, absorbed onto silica gel and
0
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5
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0
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9
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2
3
4
5
6
7
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9
0
chromatographed (50% EtOAc/DCM to DCM to 20% MeOH/DCM) to afford compound 6 as a
1
white solid (R = 0.4, 10% MeOH/EtOAc, 750 mg, 11% yield): mp 198 °C (dec); H NMR (DMSOꢀ
f
d6) δ 2.85 (t, J = 7.2 Hz, 2H), 2.92 (t, J = 7.1 Hz, 2H), 3.06 (t, J = 7.2 Hz, 2H), 3.25 (t, J = 7.2 Hz,
13
2H), 3.80 (s, 2H), 7.05 (s, 2H), 7.18–7.40 (m, 5H), 12.69 (s, 1H); C NMR (DMSOꢀd ) δ 29.4, 30.0,
6
30.1, 30.2, 41.6, 126.9, 128.5, 129.3, 134.7, 156.4, 158.6, 162.3, 168.5, 169.4. Anal. Calcd. for
C H N O S ꢃ0.25MeOH: C, 47.08; H, 4.62; N, 20.27; S, 23.20. Found: C, 47.41; H, 4.38; N,
16
18
6
1 3
19.97; S, 22.83.
Nꢀ(5ꢀ(2ꢀ(2ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ethylsulfinyl)ethyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ2ꢀ
phenylacetamide (7). To a solution of sulfide 6 (40 mg, 0.1 mmol) in MeOH (4 mL) was added
5% H O (5 drops). After 18 h, MeOH was removed. The resulting residue was purified by
3
2
2
1
preparative HPLC to obtain sulfoxide 7 as a white solid (25 mg, 60% yield): mp 181 °C (dec); H
NMR (DMSOꢀd ) δ 3.06–3.15 (m, 2H), 3.17–3.29 (m, 4H), 3.36–3.40 (m, 2H), 3.80 (s, 2H), 7.10 (s,
6
2
H), 7.26–7.35 (m, 5H), 12.74 (br s, 1H). Anal. Calcd. for C H N O S ꢃ0.8H Oꢃ0.35NaCl: C,
16 18 6 2 3 2
42.01; H, 4.32; N, 18.37; S, 21.03. Found: C, 42.39; H, 4.56; N, 18.21; S, 20.65.
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ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butanoic acid (9a). Methyl 5ꢀchloroꢀ5ꢀoxopentanoate 8a (10
mL, 72 mmol) and thiosemicarbazide (7.0 g, 72 mmol) were dissolved in POCl (25 mL) and heated
3
to 85 °C for 5 h. The reaction was cooled and the viscous oil was poured onto ice. The filtrate was
10
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basified using NaOH pellets to pH ~5. The precipitate was filtered off to obtain 9a as an offꢀwhite
1
solid (9.7 g, 72% yield): H NMR (DMSOꢀd ) δ 1.82 (quint, J = 7.6 Hz, 2H), 2.28 (t, J = 7.3 Hz,
6
2H), 2.80 (t, J = 7.6 Hz, 2H), 7.03 (br s, 2H).
4ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butanoic acid (10a). To compound 9a (4.0 g, 22
mmol) in DCM (100 mL), was added TEA (6.6 mL, 47 mmol). Next, phenylacetyl chloride (3.1 mL,
4 mmol) was added dropwise. After stirring for 4 d, the product was extracted with DCM (3 × 100
2
mL) and brine (100 mL). The organic layer was dried over MgSO and concentrated in vacuo to
4
obtain an orange oil. The crude material was purified by column chromatography (5% MeOH/DCM)
to obtain an offꢀwhite solid. The solid was triturated with EtOAc to afford 10a as a white solid (1.0
1
g, 15% yield): H NMR (DMSOꢀd ) δ 1.89 (quint, J = 7.6 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 2.98 (t,
6
J = 7.6 Hz, 2H), 3.79 (s, 2H), 7.24–7.35 (m, 5H), 12.13 (br s, 1H), 12.68 (br s, 1H).
Nꢀ(5ꢀ(3ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)propyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ2ꢀphenylacetamide
(11a). Compound 10a (600 mg, 2.0 mmol) and thiosemicarbazide (180 mg, 2.0 mmol) were
dissolved in POCl (6 mL) and heated to 85 °C for 4 h. The reaction was cooled and the viscous oil
3
was poured onto ice. The solution was basified using NaOH pellets to pH ~5. The precipitate was
filtered off and triturated with H O, followed by EtOAc to obtain 11a as a tan solid (25 mg, 4%
2
1
yield): mp 185 °C (dec); H NMR (DMSOꢀd ) δ 2.05 (quint, J = 7.3 Hz, 2H), 2.87 (t, J = 7.5 Hz,
6
13
2
H), 3.03 (t, J = 7.5 Hz, 2H), 3.81 (s, 2H), 7.04 (s, 2H), 7.26–7.36 (m, 5H); C NMR (DMSOꢀd )
6
δ 28.1, 28.6, 28.6, 41.6, 126.9, 128.4, 129.3, 134.7, 157.4, 158.3, 163.4, 168.3, 169.4. Anal. Calcd.
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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5
5
5
5
5
6
for C H N O S ꢃ0.02H Oꢃ0.1EtOAcꢃ0.06NaCl: C, 49.57; H, 4.55; N, 22.52; S, 17.19. Found: C,
1
5
16
6
1
2
2
49.81; H, 4.46; N, 22.92; S, 16.79.
5ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid (9b). Methyl adipoyl chloride 8b (25 g, 0.14
0
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9
0
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9
0
mol) and thiosemicarbazide (12.8 g, 0.14 mol) were dissolved in POCl (50 mL) and heated to 85 °C
3
for 5 h. The reaction was cooled and the viscous oil was poured onto ~100g of ice. The filtrate was
basified using 50% NaOH to pH ~5. The precipitate was filtered off to obtain 9b as a white solid (25
1
g, 89% yield): H NMR (DMSOꢀd ) δ 1.49–1.65 (m, 4H), 2.23 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 7.2
6
Hz, 2H), 7.00 (br s, 2H), 12.02 (br s, 1H).
5
ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid (10b). To compound 9b (30
g, 0.15 mol) in THF (300 mL), was added TEA (46 mL, 0.33 mol). Next, phenylacetyl chloride
22 mL, 0.16 mmol) was added via addition funnel. After stirring for 2 d, the reaction was
(
concentrated in vacuo to remove THF. The residue was triturated with hot water (~1000 mL) for
1
3
0 min and the solid was filtered to obtain 10b as a offꢀwhite solid (44 g, 92% yield): H NMR
(
DMSOꢀd ) δ 1.53 (quint, J = 7.6 Hz, 2H), 1.68 (quint, J = 7.6 Hz, 2H), 2.23 (t, J = 7.6 Hz, 2H),
6
2.96 (t, J = 7.6 Hz, 2H), 3.79 (s, 2H), 7.24–7.35 (m, 5H), 12.04 (br s, 1H), 12.67 (br s, 1H).
Nꢀ(5ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ2ꢀphenylacetamide (11b).
Compound 10b (1.2 g, 3.8 mmol) and thiosemicarbazide (0.34 g, 3.8 mmol) were dissolved in
POCl (5 mL) and heated to 85 °C for 4 h. The reaction was cooled and the viscous oil was poured
3
onto ice. The solution was basified using NaOH pellets to pH ~5. The precipitate was filtered off to
obtain offꢀwhite solid. The solid was triturated with boiling water (~100 mL), followed by hot
1
EtOAc to afford 11b as a offꢀwhite solid (0.63 g, 45% yield): mp 210 °C (dec); H NMR (DMSOꢀ
d6) δ 1.64–1.75 (m, 4H), 2.82 (t, J = 7.2 Hz, 2H), 2.99 (t, J = 7.2 Hz, 2H), 3.79 (s, 2H), 7.07 (br s,
13
2
H), 7.23–7.35 (m, 5H), 12.67 (br s, 1H); C NMR (DMSOꢀd ) δ 28.1, 28.2, 28.4, 28.9, 41.5, 126.9,
6
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28.4, 129.2, 134.6, 158.0, 158.2, 163.8, 168.2, 169.3. Anal. Calcd. for C H N O S ꢃ0.4H O: C,
16
18
6
1
2
2
5
0.35; H, 4.96; N, 22.02; S, 16.80. Found: C, 50.67; H, 4.73; N, 21.71; S, 16.40.
Methyl 5ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate (12). To a solution of carboxylic acid 9b
4.0 g, 20 mmol) in MeOH (100 mL), was added concentrated H SO (4 mL). The reaction mixture
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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(
2
4
was stirred at 65 °C for 2 d. MeOH was removed and the product was extracted with saturated
NaHCO (100 mL) and DCM (4 × 50 mL). The organic layer was dried over MgSO and
3
4
concentrated in vacuo to obtain a white residue. The residue was triturated with Et O to afford 12 as
2
1
a white solid (3.0 g, 71% yield): H NMR (DMSOꢀd ) δ 1.56–1.62 (m, 4H), 2.34 (t, J = 7.2 Hz, 2H),
6
2.79 (t, J = 7.2 Hz, 2H), 3.58 (s, 3H), 7.00 (s, 2H).
Methyl 5ꢀ(5ꢀ(3ꢀphenylpropanamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate (13a). To compound 12
(
1.0 g, 4.6 mmol) in DCM (20 mL) was added NEt (1.4 mL, 10 mmol). This was followed by
3
addition of 3ꢀphenylpropanoyl chloride (0.76 mL, 5.1 mmol) dropwise. The reaction mixture was
stirred at room temperature for 24 h. The product was extracted with DCM (3 × 50 mL) and brine
(
30 mL). The organic layer was dried over MgSO , concentrated in vacuo to obtain offꢀwhite solid.
4
1
The solid was triturated with Et O to obtain 13a as a white solid (1.5 g, 95% yield): H NMR
2
(
DMSOꢀd ) δ 1.54–1.61 (m, 2H), 1.66–1.72 (m, 2H), 2.35 (t, J = 7.3 Hz, 2H), 2.77 (t, J = 7.8 Hz,
6
2H), 2.92–2.99 (m, 4H), 3.58 (s, 3H), 7.17–7.30 (m, 5H), 12.42 (s, 1H).
Methyl 5ꢀ(5ꢀbenzamidoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate (13b). Compound 13b was prepared
from compound 12 as described for the preparation of 13a except benzoyl chloride was used in place
of 3ꢀphenylpropanoyl chloride. The crude material was purified by trituration with EtOAc: white
1
solid (48% yield); H NMR (DMSOꢀd ) δ 1.57–1.64 (m, 2H), 1.70–1.78 (m, 2H), 2.37 (t, J = 7.3 Hz,
6
2H), 3.02 (t, J = 7.3 Hz, 2H), 3.59 (s, 3H), 7.55 (t, J = 8.0 Hz, 2H), 7.66 (t, J = 7.2 Hz, 1H), 8.09 (d,
J = 7.6 Hz, 2H), 12.87 (br s, 1H).
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Methyl 5ꢀ(5ꢀ(2ꢀcyclohexylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate (13c). Compound 13c
was prepared from compound 12 as described for the preparation of 13a except 2ꢀcyclohexylacetyl
1
chloride was used in place of 3ꢀphenylpropanoyl chloride: white solid (59% yield); H NMR
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(
CDCl ) δ 1.01–1.29 (m, 5H), 1.61–1.94 (m, 10H), 2.35 (t, J = 7.2 Hz, 2H), 2.54 (d, J = 7.3 Hz, 2H),
3
3.02 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 12.45 (br s, 1H).
Methyl 5ꢀ(5ꢀ(2ꢀpꢀtolylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate (13d). Compound 13d was
prepared from compound 12 as described for the preparation of 13a except 2ꢀpꢀtolylacetyl chloride
1
was used in place of 3ꢀphenylpropanoyl chloride: white solid (86% yield); H NMR (DMSOꢀd ) δ
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1.54–1.60 (m, 2H), 1.65–1.71 (m, 2H), 2.27 (s, 3H), 2.34 (t, J = 7.2 Hz, 2H), 2.96 (t, J = 7.3 Hz,
2H), 3.57 (s, 3H), 3.74 (s, 2H), 7.13 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 12.64 (s, 1H).
Methyl 5ꢀ(5ꢀ(2ꢀ(4ꢀfluorophenyl)acetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate (13e). Compound
13e was prepared from compound 12 as described for the preparation of 13a except 2ꢀ(4ꢀ
fluorophenyl)acetyl chloride was used in place of 3ꢀphenylpropanoyl chloride: white solid (84%
1
yield); H NMR (CDCl ) δ 1.70–1.76 (m, 2H), 1.79–1.84 (m, 2H), 2.34 (t, J = 7.2 Hz, 2H), 3.04 (t, J
3
= 7.3 Hz, 2H), 3.63 (s, 3H), 3.99 (s, 2H), 7.00 (t, J = 8.7 Hz, 2H), 7.39–7.42 (dd, J = 5.4, 8.7 Hz,
2H), 13.02 (br s, 1H).
Methyl 5ꢀ(5ꢀ(2ꢀphenylpropanamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate (13f). Compound 13f
was prepared from compound 12 as described for the preparation of 13a except 2ꢀphenylpropanoyl
1
chloride was used in place of 3ꢀphenylpropanoyl chloride: white solid (74% yield); H NMR
(CDCl ) δ 1.61 (d, J = 6.8 Hz, 3H), 1.72–1.77 (m, 2H), 1.80–1.86 (m, 2H), 2.35 (t, J = 7.2 Hz, 2H),
3
3.05 (t, J = 7.3 Hz, 2H), 3.64 (s, 3H), 4.11 (q, J = 7.1 Hz, 2H), 7.26–7.32 (m, 3H), 7.42 (dd, J = 1.3,
8.1 Hz, 2H), 11.47 (br s, 1H).
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ꢀ(5ꢀ(3ꢀphenylpropanamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid (14a). To 13a (1.5 g, 4.4
mmol) in MeOH (70 mL) and H O (30 mL) was added LiOH (11 mmol). The reaction mixture was
2
stirred at 40 °C for 24 h. MeOH was removed and solution was diluted with ~20 mL H O. The
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reaction was acidified to pH ~ 2. The precipitate was collected by vacuum filtration to obtain 14a as
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a white solid (1.0 g, 69% yield): H NMR (DMSOꢀd ) δ 1.51–1.58 (t, J = 7.5 Hz, 2H), 1.66–1.74 (t,
6
J = 7.6 Hz, 2H), 2.25 (t, J = 7.4 Hz, 2H), 2.77 (t, J = 7.6 Hz, 2H), 2.90–2.98 (m, 4H), 7.16–7.30 (m,
5H), 12.05 (br s, 1H), 12.41 (br s, 1H).
5ꢀ(5ꢀbenzamidoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid (14b). Compound 14b was prepared as
described for the preparation of 14a except methyl 5ꢀ(5ꢀbenzamidoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate
1
13b was used in place of 13a: offꢀwhite solid (83% yield); H NMR (DMSOꢀd ) δ 1.58 (m, 2H),
6
1
.74 (m, 2H), 2.27 (t, J = 7.3 Hz, 2H), 3.02 (t, J = 7.5 Hz, 2H), 7.56 (t, J = 7.8 Hz, 2H), 7.66 (t, J =
.0 Hz, 1H), 8.09 (d, J = 7.1 Hz, 2H), 12.18 (br s, 1H), 12.93 (br s, 1H).
7
5ꢀ(5ꢀ(2ꢀcyclohexylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid (14c). Compound 14c was
prepared as described for the preparation of 14a except methyl 5ꢀ(5ꢀ(2ꢀcyclohexylacetamido)ꢀ1,3,4ꢀ
1
thiadiazolꢀ2ꢀyl)pentanoate 13c was used in place of 13a: white solid (79% yield); H NMR (DMSOꢀ
d6) δ 0.90–0.99 (m, 2H), 1.13–1.25 (m, 3H), 1.51–1.78 (m, 10H), 2.25 (t, J = 7.3 Hz, 2H), 2.33 (d, J
=
7.1 Hz, 2H), 2.96 (t, J = 7.3 Hz, 2H), 12.04 (br s, 1H), 12.36 (br s, 1H).
ꢀ(5ꢀ(2ꢀpꢀtolylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid (14d). Compound 14d was
5
prepared as described for the preparation of 14a except methyl 5ꢀ(5ꢀ(2ꢀpꢀtolylacetamido)ꢀ1,3,4ꢀ
1
thiadiazolꢀ2ꢀyl)pentanoate 13d was used in place of 13a: white solid (71% yield); H NMR (DMSOꢀ
d6) δ 1.49–1.57 (m, 2H), 1.64–1.72 (m, 2H), 2.24 (t, J = 7.3 Hz, 2H), 2.27 (s, 3H), 2.96 (t, J = 7.5
Hz, 2H), 3.73 (s, 2H), 7.13 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 12.04 (br s, 1H), 12.62 (br
s, 1H).
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ꢀ(5ꢀ(2ꢀ(4ꢀfluorophenyl)acetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid (14e). Compound 14e
was prepared as described for the preparation of 14a except methyl 5ꢀ(5ꢀ(2ꢀ(4ꢀ
fluorophenyl)acetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoate 13e was used in place of 13a: white solid
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(
63% yield); H NMR (DMSOꢀd ) δ 1.54 (m, 2H), 1.69 (m, 2H), 2.24 (t, J = 7.3 Hz, 2H), 2.97 (t, J =
6
7.5 Hz, 2H), 3.80 (s, 2H), 7.16 (t, J = 8.9 Hz, 2H), 7.36 (dd, J = 3.1, 8.6 Hz, 2H), 12.05 (br s, 1H),
2.66 (br s, 1H).
ꢀ(5ꢀ(2ꢀphenylpropanamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid (14f). Compound 14f was
1
5
prepared as described for the preparation of 14a except methyl 5ꢀ(5ꢀ(2ꢀphenylpropanamido)ꢀ1,3,4ꢀ
1
thiadiazolꢀ2ꢀyl)pentanoate 13f was used in place of 13a: white solid (91% yield); H NMR DMSOꢀ
d6) δ 1.44 (d, J = 7.1 Hz, 3H), 1.51–1.55 (m, 2H), 1.66–1.70 (m, 2H), 2.24 (t, J = 7.2 Hz, 2H), 2.95
(
t, J = 7.2 Hz, 2H), 4.00 (q, J = 7.1 Hz, 1H), 7.25–7.35 (m, 5H), 12.05 (br s, 1H), 12.61 (br s, 1H).
Nꢀ(5ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ3ꢀphenylpropanamide
15a). Compound 15a was prepared as described for the preparation of 11b except 5ꢀ(5ꢀ(3ꢀ
(
phenylpropanamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid 14a was used in place of 10b. The
crude material was purified by trituration with mildly basic water (pH ~ 10, 10mL): offꢀwhite
1
solid (33% yield); mp 212 °C (dec); H NMR (DMSOꢀd ) δ 1.67–1.66 (m, 4H), 2.75–2.84 (m,
6
4
H), 2.91 (t, J = 7.6 Hz, 2H), 2.99 (t, J = 7.2 Hz, 2H), 7.03 (s, 2H), 7.16–7.30 (m, 5H), 12.45 (br
1
3
s, 1H); C NMR (DMSOꢀd ) δ 28.2, 28.3, 28.4, 29.0, 30.3, 36.5, 126.1, 128.3, 128.4, 140.6,
6
1
58.0, 158.1, 163.4, 168.2, 170.6. Anal. Calcd. for C H N O Sꢃ0.7H Oꢃ0.03NaCl: C, 50.68; H,
17 20 6 2 2
5.35; N, 20.68; S, 15.92; Found: C, 51.08; H, 4.95; N, 20.44; S, 15.59.
Nꢀ(5ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)benzamide
(15b).
Compound 15b was prepared as described for the preparation of 11b except 5ꢀ(5ꢀbenzamidoꢀ
1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid 14b was used in place of 10b. The crude material was
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purified by trituration with mildly basic water (pH ~ 10, 2 × 10mL): tan solid (38% yield); mp
1
215 °C (dec); H NMR (DMSOꢀd ) δ 1.68–1.82 (m, 4H), 2.85 (t, J = 7.2 Hz, 2H), 3.04 (t, J = 7.2
6
Hz, 2H), 7.01 (s, 2H), 7.55 (t, J = 7.8 Hz, 2H), 7.65 (t, J = 7.5 Hz, 1H), 8.10 (d, J = 7.3 Hz, 2H),
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1
3
1
2.83 (br s, 1H); C NMR (DMSOꢀd ) δ 28.3, 28.3, 28.5, 29.0, 128.4, 128.6, 131.8, 132.9,
6
1
58.0, 159.4, 164.0, 165.3, 168.2. Anal. Calcd. for C H N O Sꢃ0.19EtOAcꢃ0.2NaCl: C, 48.67;
15
16
6
2
H, 4.54; N, 21.61; S,16.49; Found: C, 49.06; H, 4.52; N, 22.00; S, 16.45.
Nꢀ(5ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ2ꢀcyclohexylacetamide
(
15c). Compound 15c was prepared as described for the preparation of 11b except 5ꢀ(5ꢀ(2ꢀ
cyclohexylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid 14c was used in place of 10b. The
crude material was purified by trituration with mildly basic water (pH ~ 10, 2 × 10 mL),
1
followed by trituration with EtOAc: offꢀwhite solid (18% yield); mp 218 °C (dec); H NMR
(
DMSOꢀd ) δ 0.90–0.99 (m, 2H), 1.10–1.22 (m, 4H), 1.58–1.74 (m, 9H), 2.33 (d, J = 7.1 Hz,
6
1
3
2
H), 2.83 (t, J = 7.2 Hz, 2H), 2.99 (t, J = 7.2 Hz, 2H), 7.00 (s, 2H), 12.34 (br s, 1H); C NMR
(
DMSOꢀd ) δ 25.6, 25.7, 28.3, 28.3, 28.5, 29.0, 32.4, 34.7, 42.6, 158.0, 158.3, 163.5, 168.2,
6
1
70.7. Anal. Calcd. for C H N O S ꢃ0.53EtOAc: C, 50.94; H, 6.66; N, 19.67; S, 15.01; Found:
16 24 6 1 2
C, 51.35; H, 6.25; N, 20.03; S, 14.90.
Nꢀ(5ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ2ꢀpꢀtolylacetamide
(
15d). Compound 15d was prepared as described for the preparation of 11b except 5ꢀ(5ꢀ(2ꢀpꢀ
tolylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid 14d was used in place of 10b. The crude
material was purified by trituration with boiling water (2 × 50 mL), followed by trituration with
1
MeOH: offꢀwhite solid (57% yield); mp 225 °C (dec); H NMR (DMSOꢀd ) δ 1.64–1.75 (m,
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2H), 2.26 (s, 3H), 2.81 (t, J = 7.1 Hz, 2H), 2.98 (t, J = 7.2 Hz, 2H), 3.73 (s, 2H), 7.00 (s, 2H),
13
7
.12 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 12.63 (br s, 1H); C NMR (DMSOꢀd ) δ 20.7,
6
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8.2, 28.3, 28.4, 29.0, 41.2, 129.0, 129.1, 131.6, 136.0, 158.0, 158.2, 163.8, 168.2, 169.5. Anal.
Calcd. for C H N O S ꢃ0.35MeOHꢃ0.1H O: C, 51.90; H, 5.42; N, 20.93; S, 15.97; Found: C,
17 20
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2.23; H, 5.09; N, 20.57; S, 16.20.
Nꢀ(5ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ2ꢀ(4ꢀ
fluorophenyl)acetamide (15e). Compound 15e was prepared as described for the preparation of
1b except 5ꢀ(5ꢀ(2ꢀ(4ꢀfluorophenyl)acetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid 14e was
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used in place of 10b. The crude material was purified by trituration with mildly basic water (pH
1
~ 9, 2 × 30 mL), followed by trituration with EtOAc: tan solid (56%); mp 203 °C (dec); H NMR
(DMSOꢀd ) δ 1.66–1.73 (m, 4H), 2.81 (t, J = 7.2 Hz, 2H), 2.99 (t, J = 7.1 Hz, 2H), 3.80 (s, 2H),
6
7.03 (br s, 2H), 7.15 (t, J = 8.8 Hz, 2H), 7.33–7.37 (dd, J = 5.7, 8.5 Hz, 2H), 12.69 (br s, 1H).
Anal. Calcd. for C H N O FS ꢃ0.2EtOAcꢃ0.09H Oꢃ0.4NaCl: C, 46.38; H, 4.35; N, 19.32; S,
1
6
17
6
1
2
2
1
4.74; Found: C, 46.78; H, 4.31; N, 18.92; S, 14.36.
Nꢀ(5ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀ2ꢀphenylpropanamide
(15f). Compound 15f was prepared as described for the preparation of 11b except 5ꢀ(5ꢀ(2ꢀ
phenylpropanamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanoic acid 14f was used in place of 10b. The
crude material was purified by trituration with boiling water (~ 50 mL), followed by trituration
1
with MeOH: tan solid (40%); mp 213 °C (dec); H NMR (DMSOꢀd ) δ 1.43 (d, J = 8 Hz, 3H),
6
1.64–1.75 (m, 4H), 2.81 (t, J = 7.2 Hz, 2H), 2.97 (t, J = 7.1 Hz, 2H), 3.98 (q, J = 7.0 Hz, 1H),
1
3
7.01 (s, 2H), 7.22–7.26 (m, 1H), 7.30–7.37 (m, 4H), 12.56 (br s, 1H); C NMR (DMSOꢀd )
6
δ 18.3, 28.2, 28.3, 28.5, 29.0, 45.0, 127.0, 127.4, 128.5, 140.9, 158.0, 158.9, 163.5, 168.2, 172.7.
Anal. Calcd. for C H N O S ꢃ0.5H Oꢃ0.3MeOH: C, 51.04; H, 5.50; N, 20.64; S, 15.75; Found:
1
7
20
6
1
2
2
C, 50.86; H, 5.23; N, 20.49; S, 15.34.
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Methyl 7ꢀchloroꢀ6ꢀoxoheptanoate (16). The methyl adipoyl chloride 8b (2.5 mL, 15.3 mmol)
was dissolved in DCM (15 mL) followed by slow addition to an ethereal solution of
diazomethane (generated from 25 g of Diazald) at 0 °C. The solution was allowed to stir at 0 °C
for 1.5 h followed by quenching with 10 mL of 48% HBr (bubbling). The solution was diluted
with 40 mL DCM then put in a separatory funnel and the HBr was siphoned off. The organic
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layer was washed with 50 mL of satd NaHCO and 50 mL brine. The organic layer was
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concentrated and chromatographed in 20% EtOAc/hex with the desired product R = 0.4 (20%
f
1
EtOAc/hex, sulfuric acid charring) to afford 16 (2.9g, 98%): H NMR (CDCl ) δ 1.66 (m, 4H),
3
2.35 (m, 2H), 2.67 (m, 2H), 3.67 (s, 3H), 4.08 (s, 2H).
Methyl 5ꢀ(2ꢀaminothiazolꢀ4ꢀyl)pentanoate (17). The αꢀchloroketone 16 (2.8 g, 14.5 mmol)
was dissolved in EtOH (20 mL) followed by addition of thiourea (1.1 g, 14.5 mmol) and heated
at 60 °C for 18 hours. The solvent was removed and the crude mixture was partitioned between
DCM and satd NaHCO (20 mL). The DCM layer was dried and concentrated to afford a white
3
solid that was triturated with Et O to afford 2.5 g of the desired aminothiazole 17 (81%): mp
2
1
123–128 °C; H NMR (CDCl ) δ, 1.67 (m, 4H), 2.33 (m, 2H), 2.54 (m, 2H), 3.66 (s, 3H), 4.95
3
(br s, 2H), 6.10 (s, 1H).
Methyl 5ꢀ(2ꢀ(2ꢀphenylacetamido)thiazolꢀ4ꢀyl)pentanoate (18). The aminothiazole 17 (2.0 g,
9.35 mmol) was dissolved in DCM (30 mL) followed by addition of triethylamine (1.4 mL, 10.3
mmol) and dropwise addition of phenyl acetyl chloride (1.45 g, 9.35 mmol). The reaction was
stirred overnight then quenched with 20 mL of 1N HCl. The crude mixture was partitioned
between DCM and satd NaHCO (20 mL). The DCM layer was dried and concentrated, then
3
chromotographed on silica gel (1/1 hexanes/EtOAc) to afford 2.5 g (81%) of the desired methyl
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ester 18: H NMR (CDCl ) δ 1.64 (m, 4H), 2.31 (m, 2H), 2.61 (m, 2H), 3.65 (s, 3H), 3.81 (s,
3
2H), 6.52 (s, 1H), 7.39–7.27 (m, 5H), 8.95 (br s, 1H).
5
ꢀ(2ꢀ(2ꢀphenylacetamido)thiazolꢀ4ꢀyl)pentanoic acid (19). The methyl ester 18 (2.0 g) was
0
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7
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9
0
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7
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9
0
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dissolved in 1N NaOH (20 mL) and dioxane (10 mL) followed by stirring for 2 h at 60 °C. The
dioxane was removed and the basic solution was acidified with conc. HCl after which a white
precipitate crashed out of solution. This precipitate collected by vacuum filtration, was washed
1
with water and then triturated with ether to afford 19 (1.75 g, 92% yield): H NMR (DMSOꢀd ) δ
6
1
.48 (m, 2H), 1.61 (m, 2H), 2.22 (t, J = 7.2 Hz, 2H), 2.58 (t, J = 7.3 Hz, 2H), 3.72 (s, 2H), 6.75
(
s, 1H), 7.32 (m, 5H), 12.20 (br s, 1H), 12.25 (br s, 1H).
Nꢀ(4ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)thiazolꢀ2ꢀyl)ꢀ2ꢀphenylacetamide
(20).
Compound 20 was prepared as described for the preparation of 11b except 5ꢀ(2ꢀ(2ꢀ
phenylacetamido)thiazolꢀ4ꢀyl)pentanoic acid 19 was used in place of 10b: offꢀwhite solid (29%
1
yield); mp 192–197 °C; H NMR (DMSOꢀd ) δ 1.63–1.65 (m, 4H), 2.61 (t, J = 6.8 Hz, 2H), 2.80
6
1
3
(
t, J = 6.8 Hz, 2H), 3.72 (s, 2H), 6.75 (s, 1H), 7.00 (s, 2H), 7.25–7.33 (m, 5H), 12.32 (s, 1H); C
NMR (DMSOꢀd ) δ 27.8, 28.5, 29.2, 30.4, 41.7, 107.4, 126.8, 128.4, 129.2, 135.0, 151.0, 157.3,
6
1
58.2, 168.1, 169.0. Anal. Calcd. for C H N O S ꢃ0.5H Oꢃ0.02EtOAc: C, 53.39; H, 5.29; N,
17 19 5 1 2 2
18.22; S, 16.69. Found: C, 53.75; H, 5.11; N, 17.88; S, 16.31.
Ethyl 7ꢀoxoheptanoate (22). Compound 22 was prepared from acid chloride 21 as previously
2
2
1
described . The aldehyde 22 was used without further purification: red oil (95% yield); H
NMR (CDCl ) δ 1.22 (t, J = 7.2 Hz, 3H), 1.32–1.40 (m, 2H), 1.58–1.66 (m, 4H), 2.28 (t, J = 7.5
3
Hz, 2H), 2.45 (tt, J = 2.2, 7.3 Hz, 2H), 4.08 (q, J = 7.1 Hz, 2H), 9.74 (t, J = 1.8 Hz, 1H).
Ethyl 6ꢀbromoꢀ7ꢀoxoheptanoate (23). Compound 23 was prepared from aldehyde 22 as was
2
2
previously described. The bromide 23 was used without further purification: orange oil (62%
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yield); H NMR (CDCl ) δ 1.24 (t, J = 7.2 Hz, 3H) 1.62–1.75 (m, 4H), 2.28 (t, J = 7.5 Hz, 1H),
3
2
.34 (t, J = 7.1 Hz, 2H), 2.39–2.43 (m, 2H), 4.12 (q, J = 7.1 Hz, 2H), 9.20 (s, 1H).
Ethyl 5ꢀ(2ꢀaminothiazolꢀ5ꢀyl)pentanoate (24). Compound 24 was prepared from αꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
27
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60
2
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bromoaldehyde 23 as was previously described. The crude material was purified by column
1
chromatography (99.9% EtOAc, 0.1% NH OH): brown oil (31% yield); H NMR (CDCl ) δ 1.22
4
3
(
t, J = 7.2 Hz, 3H), 1.54–1.69 (m, 4H), 2.32 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 6.8 Hz, 2H), 4.08 (q,
J = 7.2 Hz, 2H), 4.85 (br s, 2H), 6.70 (s, 1H).
Ethyl 5ꢀ(2ꢀ(2ꢀphenylacetamido)thiazolꢀ5ꢀyl)pentanoate (25). Compound 25 was prepared as
described for the preparation of 18 except ethyl 5ꢀ(2ꢀaminothiazolꢀ5ꢀyl)pentanoate 24 was used
1
in place of 17: white powder (70% yield); H NMR (CDCl ) δ 1.22 (t, J = 7.1 Hz, 3H) 1.63–1.67
3
(
m, 4H), 2.27–2.30 (m, 2H), 2.72–2.75 (m, 2H), 3.80 (s, 2H), 4.09 (q, J = 7.1 Hz, 2H), 7.03 (s,
1H), 7.28–7.38 (m, 5H), 9.66 (br s, 1H).
5ꢀ(2ꢀ(2ꢀphenylacetamido)thiazolꢀ5ꢀyl)pentanoic acid (26). Compound 26 was prepared as
described for the preparation of 19 except ethyl 5ꢀ(2ꢀ(2ꢀphenylacetamido)thiazolꢀ5ꢀyl)pentanoate
1
2
5 was used in place of 18: white powder (91% yield); H NMR (DMSOꢀd ) δ 1.50–1.56 (m,
6
4H), 2.22 (t, J = 6.9 Hz, 2H), 2.71 (t, J = 6.9 Hz, 2H), 3.72 (s, 2H), 7.16 (s, 1H), 7.23–7.35 (m,
5H), 12.02 (br s, 1H), 12.18 (br s, 1H).
Nꢀ(5ꢀ(4ꢀ(5ꢀaminoꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)butyl)thiazolꢀ2ꢀyl)ꢀ2ꢀphenylacetamide
(27).
Compound 27 was prepared as described for the preparation of 11b except 5ꢀ(2ꢀ(2ꢀ
phenylacetamido)thiazolꢀ5ꢀyl)pentanoic acid 26 was used in place of 10b: offꢀwhite solid (55%
1
yield); H NMR (DMSOꢀd ) δ 1.63–1.65 (m, 4H), 2.61 (t, J = 6.7 Hz, 2H), 2.80 (t, J = 6.8 Hz,
6
2
H), 3.72 (s, 2H), 6.75 (s, 1H), 7.00 (br s, 2H), 7.24–7.33 (m, 5H), 12.02 (br s, 1H), 12.18 (br s,
1
3
1
H); C NMR (DMSOꢀd ) δ 27.8, 28.5, 29.1, 30.4, 41.7, 107.4, 126.8, 128.4, 129.2, 135.0,
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51.0, 157.3, 158.2, 168.1, 169.0. Anal. Calcd. for C H N O S ꢃ0.01H Oꢃ0.27MeOH: C, 54.25;
17
19
5
1
2
2
H, 5.30; N, 18.32; S, 16.77. Found: C, 54.49; H, 5.05; N, 17.97; S, 16.38.
ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀNꢀ(2ꢀ(pyrrolidinꢀ1ꢀyl)ethyl)pentanamide
29a). To carboxylic acid 10b (2.5 g, 7.9 mmol) dissolved in DMF was added DIEA (5.5 mL, 32
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(
mmol). The reaction mixture was cooled to 0 °C. HATU (3.0 g, 7.9 mmol) was added and stirred
for 10 minutes, followed by addition of 2ꢀ(pyrrolidinꢀ1ꢀyl)ethanamine 28a (1.1 mL, 8.6 mmol).
After 24 hours, the DMF was removed. The product was extracted with DCM (4 × 50 mL) and
washed with saturated NaHCO (50 mL). The organic layer was dried over MgSO and
3
4
concentrated to obtain offꢀwhite solid. The solid was triturated with EtOAc to obtain 29a as an
1
offꢀwhite solid (2.1 g, 65%): mp 152 °C (dec); H NMR (DMSOꢀd ) δ 1.52 (quint, J = 3 Hz,
6
2
H), 1.61–1.68 (m, 6H), 2.08 (t, J = 7.2 Hz, 2H), 2.44–2.48 (m, 6H), 2.95 (t, J = 7.5 Hz, 2H),
.14 (q, J = 6.6 Hz, 2H), 3.79 (s, 2H), 7.24–7.35 (m, 5H), 7.79 (t, J = 5.4 Hz, 1H), 12.28 (br s,
3
1
3
1H); C NMR (DMSOꢀd ) δ 23.0, 24.6, 28.6, 28.7, 34.5, 37.6, 41.6, 53.6, 54.9, 126.9, 128.4,
6
1
29.3, 134.7, 158.2, 164.0, 169.3, 171.7. Anal. Calcd. for C H N O S ꢃ0.15H Oꢃ0.2NaCl: C,
21 29 5 2 1 2
5
8.67; H, 6.87; N, 16.29; S, 7.46. Found: C, 58.28; H, 6.74; N, 16.26; S, 7.30.
Nꢀmethylꢀ5ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀNꢀ(2ꢀ(pyrrolidinꢀ1ꢀ
yl)ethyl)pentanamide (29b). Amide 29b was prepared from 10b as described for the
preparation of 29a except Nꢀmethylꢀ2ꢀ(pyrrolidinꢀ1ꢀyl)ethanamine 28b was used in place of 2ꢀ
(pyrrolidinꢀ1ꢀyl)ethanamine 28a. The crude material was purified using column chromatography
1
(5% MeOH, 94% DCM, 1% NH OH) to obtain 28a as an orange oil (50% yield): H NMR
4
(MeOD) δ 1.65–1.71 (m, 2H), 1.78–1.85 (m, 6H), 2.40–2.45 (m, 2H), 2.59 (m, 1H), 2.67–2.77
(m, 5H), 2.89–2.91 (m, 1H), 3.02–3.06 (m, 4H), 3.46–3.50 (m, 1H), 3.53–3.56 (m, 1H), 3.81 (s,
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H), 7.25–7.33 (m, 5H). Anal. Calcd. for C H N O S ꢃ0.3H Oꢃ0.2NaCl: C, 58.55; H, 7.28; N,
22
31
5
2
1
2
1
5.24; S, 6.98. Found: C, 58.17; H, 7.11; N, 15.31; S, 7.12.
Nꢀ(2ꢀ(1ꢀmethylpyrrolidinꢀ2ꢀyl)ethyl)ꢀ5ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
10
11
12
13
14
15
16
17
18
19
20
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22
23
24
25
26
27
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59
60
yl)pentanamide (29c). Amide 29c was prepared from 10b as described for the preparation of
29a except 2ꢀ(1ꢀmethylpyrrolidinꢀ2ꢀyl)ethanamine 28c was used in place of 2ꢀ(pyrrolidinꢀ1ꢀ
1
yl)ethanamine 28a: tan solid (30% yield); mp 121–131 °C; H NMR (DMSOꢀd ) δ 1.25–1.37
6
(m, 2H), 1.52–1.70 (m, 7H), 1.85–1.90 (m, 1H), 2.01–2.08 (m, 4H), 2.18 (s, 3H), 2.89–2.97 (m,
3H), 2.99–3.06 (m, 2H), 3.79 (s, 2H), 7.23–7.35 (m, 5H), 7.79 (t, J = 5.6 Hz, 1H), 12.47 (br s,
13
1
H); C NMR (DMSOꢀd ) δ 21.5, 24.6, 28.6, 28.7, 30.0, 32.8, 34.9, 35.9, 40.0, 41.6, 56.5, 63.7,
6
1
26.9, 128.5, 129.3, 134.7, 158.2, 164.0, 169.3, 171.5. Anal. Calcd. for C H N O S ꢃ0.45H O:
22 31
5
2
1
2
C, 60.37; H, 7.35; N, 16.00; S, 7.33. Found: C, 59.98; H, 7.11; N, 15.65; S, 7.17.
ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀNꢀ(2ꢀ(piperidinꢀ1ꢀyl)ethyl)pentanamide
9d). Amide 29d was prepared from 10b as described for the preparation of 29a except 2ꢀ
5
2
(piperidinꢀ1ꢀyl)ethanamine 28d was used in place of 2ꢀ(pyrrolidinꢀ1ꢀyl)ethanamine 28a: white
1
solid (39% yield); mp 132 °C (dec); H NMR (DMSOꢀd ) δ 1.32–1.35 (m, 2H), 1.41–1.46 (m,
6
4H), 1.49–1.56 (m, 2H), 1.58–1.64 (m, 2H), 1.61–1.68 (m, 2H), 2.07 (t , J = 7.1 Hz, 2H), 2.25–
2.30 (m, 6H), 2.95 (t , J = 7.3 Hz, 2H), 3.12 (q, J = 6.8 Hz, 2H), 3.79 (s, 2H), 7.26–7.35 (m, 5
1
3
H), 7.70 (t, J = 5.6 Hz, 1H), 12.61 (br s, 1H); C NMR (DMSOꢀd ) δ 24.0, 24.6, 25.5, 28.6,
6
28.7, 34.9, 36.1, 41.6, 54.0, 57.9, 126.9, 128.4, 129.3, 134.7, 158.2, 164.0, 169.3, 171.7. Anal.
Calcd. for C H N O Sꢃ0.12H O: C, 61.2; H, 7.29; N, 16.22; S, 7.43. Found: C, 60.80; H, 7.09;
22
31
5
2
2
N, 16.21; S, 7.52.
Nꢀ(2ꢀ(4ꢀhydroxypiperidinꢀ1ꢀyl)ethyl)ꢀ5ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
yl)pentanamide (29e). Amide 29e was prepared from 10b as described for the preparation of
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9a except 1ꢀ(2ꢀaminoethyl)piperidinꢀ4ꢀol 28e was used in place of 2ꢀ(pyrrolidinꢀ1ꢀ
1
yl)ethanamine 28a: white solid (15% yield); mp 94–99 °C; H NMR (DMSOꢀd ) δ 1.38 (m, 2H),
6
1
.50–1.56 (m, 2H), 1.61–1.68 (m, 4H), 2.08 (t, J = 7.2 Hz, 3H), 2.33–2.34 (m, 2H), 2.67–2.72
m, 2H), 2.95 (t, J = 7.1 Hz, 3H), 3.13 (m, 2H), 3.43 (m, 1H), 3.79 (s, 2H), 4.57 (br s, 1H), 7.24–
7.35 (m, 5H), 7.75 (br s, 1H), 12.67 (br s, 1H). Anal. Calcd. for C H N O Sꢃ0.25H Oꢃ0.3NaCl:
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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7
8
9
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8
9
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2
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4
5
6
7
8
9
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(
22
31
5
3
2
C, 56.51; H, 6.79 ; N, 14.98; S, 6.86. Found: C, 56.27; H, 6.84; N, 14.93; S, 6.94.
ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)ꢀNꢀ(2ꢀ(4ꢀphenylpiperidinꢀ1ꢀ
5
yl)ethyl)pentanamide (29f). Amide 29f was prepared from 10b as described for the preparation
of 29a except 2ꢀ(4ꢀphenylpiperidinꢀ1ꢀyl)ethanamine 28f was used in place of 2ꢀ(pyrrolidinꢀ1ꢀ
1
yl)ethanamine 28a: offꢀwhite solid (18% yield); mp 123–126 °C; H NMR (DMSOꢀd ) δ 1.33–
6
1.43 (m, 1H), 1.49–1.56 (m, 3H), 1.62–1.67 (m, 3H), 1.74–1.77 (m, 1H), 1.90–1.98 (m, 2H),
2.08 (t, J = 7.2 Hz, 2H), 2.34 (t, J = 6.3 Hz, 2H), 2.64–2.70 (m, 1H), 2.83–2.86 (m, 2H), 2.95 (t,
J = 7.3 Hz, 2H), 3.15 (q, J = 6.7 Hz, 2H), 3.78 (s, 2H), 7.16–7.35 (m, 10H), 7.73 (t, J = 5.7 Hz,
13
1
H), 12.66 (br s, 1H); C NMR (DMSOꢀd ) δ 24.6, 25.2, 28.6, 31.2, 34.9, 36.1, 41.5, 42.1, 53.2,
6
57.4, 126.1, 126.9, 127.1, 128.2, 128.4, 129.2, 134.6, 144.6, 158.1, 164.0, 169.3, 171.6. Anal.
Calcd. for C H N O Sꢃ0.22H Oꢃ0.08NaCl: C, 65.39; H, 6.95; N, 13.62. Found: C, 64.94; H,
2
8
35
5
2
2
6
.81; N, 14.10.
Nꢀ(2ꢀaminoethyl)ꢀ5ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanamide (29g). To
synthesize 29g, tertꢀbutyl 2ꢀ(5ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
yl)pentanamido)ethylcarbamate was first prepared from 10b as described for the preparation of
2
9a except tertꢀbutyl 2ꢀaminoethylcarbamate was used in place of 2ꢀ(pyrrolidinꢀ1ꢀyl)ethanamine
8a. To the solution of 2ꢀ(5ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀ
2
yl)pentanamido)ethylcarbamate (100 mg, 0.22 mmol) in DCM (2 mL) was added TFA (0.5 mL).
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The reaction mixture was stirred at room temperature for 2 h. H O (20 mL) was added and
2
organic layer was separated. The aqueous layer was washed with DCM (3 × 20 mL) and
concentrated to obtain white solid. The solid was triturated with Et O to obtain 95 mg of 29g as
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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35
36
37
38
39
40
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43
44
45
46
47
48
49
50
51
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53
54
55
56
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58
59
60
1
a white solid (92% yield): mp 167–172 °C; H NMR (DMSOꢀd ) δ 1.54 (quint, J = 7.6 Hz, 2H),
6
1.67 (quint, J = 7.5 Hz, 2H), 2.13 (t, J = 7.3 Hz, 2H), 2.83 (m, 2H), 2.96 (t, J = 7.2 Hz, 2H), 3.25
(
q, J = 6.0 Hz, 2H), 3.80 (s, 2H), 7.26–7.34 (m, 5H), 7.76 (br s, 3H), 8.01 (br s, 1H), 12.69 (s,
1
3
1H); C NMR (DMSOꢀd ) δ 24.2, 28.6, 28.7, 34.8, 36.4, 38.7, 41.5, 126.9, 128.4, 129.2, 134.6,
6
1
58.1, 164.0, 169.3, 172.7. Anal. Calcd. for C H N O S ꢃTFAꢃ0.2H O: C, 47.73; H, 4.93; N,
17 23 5 1 2 2
1
4.65; S, 6.71. Found: C, 47.38; H, 5.04; N, 14.50; S, 6.91.
(2ꢀ(diethylamino)ethyl)ꢀ5ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanamide
29h). Amide 29h was prepared from 10b as described for the preparation of 29a except N, Nꢀ
(
diethylethaneꢀ1,2ꢀdiamine 28h was used in place of 2ꢀ(pyrrolidinꢀ1ꢀyl)ethanamine 28a: white
1
solid (43% yield); mp 126–132 °C; H NMR (DMSOꢀd ) δ 0.91(t, J = 7.1 Hz, 6H), 1.52 (quint, J
6
=
7.6 Hz, 2H), 1.65 (quint, J = 7.6 Hz, 2H), 2.08 (t, J = 7.2 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H),
2.44 (q, J = 7.1 Hz, 4H), 2.95 (t, J = 7.4 Hz, 2H), 3.07 (q, J = 6.1 Hz, 2H), 3.79 (s, 2H), 7.24–
13
7
.35 (m, 5H), 7.69 (t, J = 5.7 Hz, 1H), 12.62 (br s, 1H); C NMR (DMSOꢀd ) δ 11.7, 24.6, 28.6,
6
28.6, 34.9, 36.8, 41.6, 46.6, 51.7, 126.9, 128.4, 129.2, 134.7, 158.2, 163.9, 169.3, 171.6. Anal.
Calcd. for C H N O S : C, 60.40; H, 7.48; N, 16.77; S, 7.68. Found: C, 60.15; H, 7.43; N,
2
1
31
5
1 2
1
6.66; S, 7.92.
Nꢀtertꢀbutylꢀ5ꢀ(5ꢀ(2ꢀphenylacetamido)ꢀ1,3,4ꢀthiadiazolꢀ2ꢀyl)pentanamide (29i). Amide 29i
was prepared from 10b as described for the preparation of 29a except 2ꢀmethylpropanꢀ2ꢀamine
2
8i was used in place of 2ꢀ(pyrrolidinꢀ1ꢀyl)ethanamine 28a. The crude material was triturated
1
with DCM and Et O: white solid (41% yield), mp 158–160 °C; H NMR (DMSOꢀd ) δ 1.21 (s,
2
6
29
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