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

Abstract

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, 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.

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 genes.¹ Kidney-type glutaminase (GLS) is widely distributed throughout extra-hepatic tissues whereas liver-type 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 growing malignant cells.^{2, 3} Recent studies have shown that Myc, an oncogenic transcription factor, increases glutamine metabolism by stimulating the expression of glutaminase directly or via suppression of miR23a and miR23b.⁴ It is conceivable that, under hypoxic conditions, glutaminolysis provides the dominant means for cancer cell metabolism.⁴ 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 medium.⁴ These findings indicate that small molecule GLS inhibitors might exert antiproliferative effects against Myc-dependent 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 glutamine-glutamate 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 HIV1-associated dementia⁵ and multiple sclerosis.⁶ 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 consequences. Indeed, recent studies showed that GLS knockouts die shortly after birth.⁷ However, mice heterozygous for the GLS (GLS hets) are fully viable despite a mild yet significant reduction in glutamate levels in some tissues.⁷ 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 conventional GLS inhibitor 1, known as 6-diazo-5-oxo-L-norleucine (DON),⁸ 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.

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 compound 2 are being explored as a new class of GLS inhibitors for the treatment of cancer.^{9, 10} Compound 3, known as bis-2-[5-(phenylacetamido)-1,3,4-thiadiazol-2-yl]ethyl sulfide or BPTES, represents another class of GLS inhibitors structurally distinct from 1 (Figure 1).¹¹ Compound 3 offers some fundamental advantages over compounds 1 and 2. Unlike 1, compound 3 does not contain any reactive chemical group that might form a covalent bond and is unlikely 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 inhibits GLS over GLS2 and γ-glutamyl transpeptidase.¹² A substrate concentration-dependent kinetic study revealed that 3 inhibits GLS in a uncompetitive manner by facilitating the formation of an inactive tetramer.^{12, 13} In addition compound 3 is capable of inhibiting the phosphate-activated GLS while compound 2 shows little ability to inhibit GLS in the presence of phosphate.¹⁰ Recently, three independent groups solved crystal structures of GLS.^14–16 Some of these structures were determined as complexes with compound 3,^{15, 16} 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.

Figure 1.

Structures of representative GLS inhibitors

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 inhibition.^17–19 Compound 3, however, is highly hydrophobic with an aqueous solubility of less than 1 μg/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.

Herein is described systematic structure-activity relationship (SAR) analysis of analogs derived from 3 as a part of our ongoing efforts to identify drug-like 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 bis-aminothiadiazole 5.^{11, 16} 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.

Scheme 1.

Synthesis of 3 and truncated analogs 5, 6 and 7^a

^a Reagents and conditions: (a) thiosemicarbazide, POCl₃, 90 °C, 79%; (b) 2.0 equiv. phenylacetyl chloride, DMA, TEA 88%; (c) 1.0 equiv. phenylacetyl chloride, DMA, TEA, 11%; (d) H₂O₂, MeOH, 60%.

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 work-up provided aminothiadiazole-containing 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.

Scheme 2.

Synthesis of 11a–b and 15a–f^a

^a Reagents and conditions: (a) thiosemicarbazide, POCl₃, 85 °C, 72% for 9a and 89% for 9b; (b) phenylacetyl chloride, TEA, THF, 15% for 10a and 92% for 10b; (c) thiosemicarbazide, POCl₃, 85 °C, 4% for 11a and 45% for 11b; (d) H₂SO₄, MeOH, 65 °C, 71%; (e) acid chloride, TEA, DCM, 48–95%; (f) LiOH, MeOH, H₂O, 40 °C, 63–91%; (g) thiosemicarbazide, POCl₃, 85 °C, 18–57%.

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.

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 afford an intermediate α-chloroketone 16,²⁰ which was reacted with thiourea to form 17.²¹ The aminothiazole 17 was converted into the desired compound 20 by acetylation, hydrolysis and cyclization. Compound 27, another thiazole-containing analog, was synthesized from acid chloride 21 as shown in Scheme 4. Rosenmund reduction of 21 gave aldehyde 22, which was brominated to afford α-bromoaldehyde 23.²² 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.

Scheme 3.

Synthesis of 20^a

^a Reagents and conditions: (a) CH₂N₂, Et₂O, DCM, 0 °C, 98%; (b) thiourea, EtOH, 60 °C, 81%; (c) phenylacetyl chloride, TEA, DCM, 81%; (d) 1N NaOH, dioxane, 60 °C, 92%; (e) thiosemicarbazide, POCl₃, 85 °C, 29%.

Scheme 4.

Synthesis of 27^a

^b Reagents and conditions: (a) H₂, 10% Pd/C, 2,6-lutidine, THF, 95%; (b) Br₂, CHCl₃, 62%; (c) thiourea, EtOH, 70 °C, 31%; (d) phenylacetyl chloride, TEA, DCM, 70%; (e) 1M NaOH, dioxane, 60 °C, 91%; (f) thiosemicarbazide, POCl₃, 85 °C, 55%.

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.

Scheme 5.

Synthesis of amides 29a–m^a

^a Reagents and conditions: (a) HATU, DMF, 0 °C to rt, 15–72%.

RESULTS AND DISCUSSION

All new synthetic compounds were tested for their ability to inhibit GLS using L-[³H]-glutamine as substrate and human kidney-type glutaminase (hKGA^124–669). Table 1 summarizes 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 (50-fold increase in IC₅₀ value from 3 to 5) has been previously reported.¹⁶ Removal of one phenylacetyl group 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 fashion.^{15, 16} 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 (Figure 2A) and 5 (Figure 2B) bind to the same allosteric site of GLS.¹⁶ 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_i value of 2.0 μM. This is consistent with the mode of inhibition reported for compound 3¹³ 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 HIV-1 protease which forms a symmetrical dimer and yet accommodates both symmetric and asymmetric inhibitors. Nevertheless, this SAR trend presented a promising opportunity to identify GLS inhibitors of lower molecular weight and hydrophobicity.

Table 1.

Inhibition of GLS by truncated derivatives of compound 3

Compd	Structure	IC50 (μM)a
3		3.3 ± 0.7
5		100
6		2.7 ± 1.7
7		61 ± 6
11a		15 ± 6
11b		1.9 ± 0.5
15a		>100
15b		7.2 ± 0.7
15c		22 ± 16
15d		4.5 ± 0.7
15e		2.6 ± 0.3
15f		12 ± 3
20		>100
27		>100

^aValues are the means ± SD of three or more independent experiments.

Figure 2.

Allosteric sites of GLS in complex with (A) compound 3 (3VOZ) and (B) compound 5 (3VP2). Compounds 3 and 5 are shown in green and cyan, respectively. Two loops at the allosteric site (one from each GLS unit) are shown in orange and magenta, respectively. Key hydrogen-bonding interactions are shown as gray dashed lines.

Figure 3.

Double-reciprocal plot of the hydrolysis of glutamine by GLS1 in the presence of compound 6. The straight lines represent the least squares fit of the data obtained by plotting the reciprocal of glutaminase activity versus the reciprocal of the glutamine concentrations.

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 IC₅₀ value of 61 OM. In the next set of SAR studies, we tested analogs 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 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′-butane-1,4-diylbis(1,3,4-thiadiazol- 2-amine), another 4-carbon linker containing GLS inhibitor (3VP4).¹⁶ 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 IC₅₀ values ranging from 2.6 to 12 OM. Only modest loss of 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 pi-stacking interactions with any aromatic residues of GLS (Figure 2A).¹⁶ 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 1,3-thiazole ring. Neither compounds inhibited GLS up to 100 OM. The complete loss of potency seen in compound 27 particularly highlights the crucial role played by N₄ of the thiadiazole ring most likely through its interaction with Phe322¹⁵ and/or Tyr394¹⁶ residue(s). Indeed, mutant forms of GLS (F322S, Y394I, or Y394L) exhibited similar enzymatic activity compared to the wild-type form but showed significantly reduced sensitivity to 3,^{15, 16} 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 μM 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 tert-butyl 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.

Table 2.

Structure activity relationships of amides 29a–m

Compd	NR1R2	IC50 (μM)a
29a		4.6 ± 1.2
29b		>100
29c		8.1 ± 2.4
29d		10 ± 1
29e		3.8 ± 0.6
29f		1.4 ± 0.3
29g		>100
29h		26 ± 6
29i		11 ± 1
29j		5.2 ± 0.9
29k		>100
29l		48 ± 13
29m		>100

^aValues are the means ± SD of three or more independent experiments.

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. Using the LC/MS based shake-flask method,²³ aqueous solubility of 3, 6, 11b and 29a was determined to be 0.144, 13, 3.4 and 683 μg/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.

Subsequent pharmacological characterization was conducted with one of the truncated analogs, compound 6. At concentrations up to 100 OM, compound 6 showed no inhibition of mouse liver-type glutaminase (mouse GLS2), whose sequence is highly homologous to that of a human GLS2.²⁴ 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, non-sulfide 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 over-expressed in the absence of tetracycline.²⁵ As shown in Figure 4A, compound 6 attenuated the growth of P493 cells in vitro in a dose-dependent manner with an IC₅₀ value near 20 OM. Previous studies have shown that similar antiproliferative effects can be achieved by GLS siRNA,⁴ glutamine deprivation,⁴ or treatment with compound 3.¹⁸ In the same assay, compound 29a inhibited the growth with IC₅₀ value near 40 μM. An attempt to assess antiproliferative activity of compound 11b was unsuccessful due to its poor aqueous solubility.

Figure 4.

Anti-proliferative effects of GLS inhibitor 6. (A) Effect of GLS inhibitor 6 on aerobic P493 human B cell growth. Error bars represent ± SD (n = 3). (B) Antitumor effect of GLS inhibitor 6 (200 Og/mice, ~12.5 mg/kg, every other day, i.p.) on P493 human B cell xenograft transplanted into SCID mice. Error bars represent ± SEM (n = 4, *p < 0.05 compared to the vehicle-treated control).

Compound 6 was also tested in an animal model in which compound 3 was previously reported to exhibit antiproliferative efficacy.¹⁸ 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 xenograft-bearing SCID mice treated with compound 6 showed significantly diminished tumor growth compared to the vehicle-treated mice. The degree of efficacy achieved by compound 6 is comparable with that of compound 3 reported previously.¹⁸ 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 non-sulfide GLS inhibitors such as 11b and 29a to determine the effects of improved metabolic stability on the degree of efficacy.

CONCLUSIONS

While kidney-type 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 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 anti-proliferative 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 xenograft-bearing 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.

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 multi-wavelength detector using a Phenomenex Luna 5 μm 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 Mel-Temp apparatus and are uncorrected. ¹H NMR spectra were recorded at 400 MHz. ¹³C NMR 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).

N,N′-(5,5′-(2,2′-thiobis(ethane-2,1-diyl))bis(1,3,4-thiadiazole-5,2-diyl))bis(2-phenylacetamide) (3)

The preparation of 3 from bisthiadiazole 5 has already been reported.^{11, 16} 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 NEt₃ (3.8 mL, 27.5 mmol) and phenylacetyl chloride (3.2 mL, 25 mmol). The reaction was stirred overnight at room temperature. DMA was removed in vacuo and the resulting solid was triturated with 10% NaHCO₃ and boiling MeOH to obtain 3 as a white solid (5.8 g, 88% yield): mp 231–234 °C; ¹H NMR (DMSO-d₆) δ 2.92 (t, J = 7.2 Hz, 4H), 3.24 (t, J = 7.1 Hz, 4H), 3.79 (s, 4H), 7.23–7.34 (m, 10H), 12.69 (s, 2H); ¹³C NMR (DMSO-d₆) δ 29.4, 30.1, 41.6, 126.9, 128.5, 129.3, 134.7, 158.6, 162.3, 169.4. Anal. Calcd. for C₂₄H₂₄N₆O₂S₃·0.5H₂O: C, 54.01; H, 4.72; N, 15.75; S, 18.02. Found: C, 54.00; H, 4.54; N, 16.0; S, 18.41.

Bis-[2-(5-Amino-[1,3,4]thiadiazol-2-yl)-ethylsulfide] (5)

The preparation of compound 5 from thiodipropionic acid 4 has already been reported.¹⁶ We used a similar procedure to synthesize 5. Thiodipropionic acid 4 (10 g, 56 mmol) and thiosemicarbazide (10 g, 56 mmol) were dissolved in POCl₃ (30 mL) and heated to 90 °C for 3 h. The reaction was cooled and the viscous oil was poured 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 H₂O. The solid was then resuspended in boiling MeOH and filtered hot. This final filtration led to desired bis-aminothiadiazole 5 as a white solid (12.7 g, 79% yield): mp 222 °C (dec); ¹H NMR (DMSO-d₆) δ 2.84 (t, J = 7.2 Hz, 4H), 3.06 (t, J = 7.2 Hz, 4H), 7.04 (s, 4H); ¹³C NMR (DMSO-d₆) δ 30.0, 30.3, 156.4, 168.5. Anal. Calcd. for C₈H₁₂N₆S₃·0.1H₂O·0.02MeOH: C, 33.12; H, 4.26; N, 28.89; S, 33.07. Found: C, 33.49; H, 4.11; N, 28.51; S, 32.84.

N-(5-{2-[2-(5-Amino-[1,3,4]thiadiazol-2-yl)-ethylsulfanyl]-ethyl}-[1,3,4]thiadiazol-2-yl)-2-phenyl-acetamide (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 chromatographed (50% EtOAc/DCM to DCM to 20% MeOH/DCM) to afford compound 6 as a white solid (R_f = 0.4, 10% MeOH/EtOAc, 750 mg, 11% yield): mp 198 °C (dec); ¹H NMR (DMSO-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, 2H), 3.80 (s, 2H), 7.05 (s, 2H), 7.18–7.40 (m, 5H), 12.69 (s, 1H); ¹³C NMR (DMSO-d₆) δ 29.4, 30.0, 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 C₁₆H₁₈N₆O₁S₃·0.25MeOH: C, 47.08; H, 4.62; N, 20.27; S, 23.20. Found: C, 47.41; H, 4.38; N, 19.97; S, 22.83.

N-(5-(2-(2-(5-amino-1,3,4-thiadiazol-2-yl)ethylsulfinyl)ethyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (7)

To a solution of sulfide 6 (40 mg, 0.1 mmol) in MeOH (4 mL) was added 35% H₂O₂ (5 drops). After 18 h, MeOH was removed. The resulting residue was purified by preparative HPLC to obtain sulfoxide 7 as a white solid (25 mg, 60% yield): mp 181 °C (dec); ¹H NMR (DMSO-d₆) 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, 2H), 7.26–7.35 (m, 5H), 12.74 (br s, 1H). Anal. Calcd. for C₁₆H₁₈N₆O₂S₃·0.8H₂O·0.35NaCl: C, 42.01; H, 4.32; N, 18.37; S, 21.03. Found: C, 42.39; H, 4.56; N, 18.21; S, 20.65.

4-(5-amino-1,3,4-thiadiazol-2-yl)butanoic acid (9a)

Methyl 5-chloro-5-oxopentanoate 8a (10 mL, 72 mmol) and thiosemicarbazide (7.0 g, 72 mmol) were dissolved in POCl₃ (25 mL) and heated to 85 °C for 5 h. The reaction was cooled and the viscous oil was poured onto ice. The filtrate was basified using NaOH pellets to pH ~5. The precipitate was filtered off to obtain 9a as an off-white solid (9.7 g, 72% yield): ¹H NMR (DMSO-d₆) δ 1.82 (quint, J = 7.6 Hz, 2H), 2.28 (t, J = 7.3 Hz, 2H), 2.80 (t, J = 7.6 Hz, 2H), 7.03 (br s, 2H).

4-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)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, 24 mmol) was added dropwise. After stirring for 4 d, the product was extracted with DCM (3 × 100 mL) and brine (100 mL). The organic layer was dried over MgSO₄ and concentrated in vacuo to obtain an orange oil. The crude material was purified by column chromatography (5% MeOH/DCM) to obtain an off-white solid. The solid was triturated with EtOAc to afford 10a as a white solid (1.0 g, 15% yield): ¹H NMR (DMSO-d₆) δ 1.89 (quint, J = 7.6 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 2.98 (t, 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).

N-(5-(3-(5-amino-1,3,4-thiadiazol-2-yl)propyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (11a)

Compound 10a (600 mg, 2.0 mmol) and thiosemicarbazide (180 mg, 2.0 mmol) were dissolved in POCl₃ (6 mL) and heated to 85 °C for 4 h. The reaction was cooled and the viscous oil was poured onto ice. The solution was basified using NaOH pellets to pH ~5. The precipitate was filtered off and triturated with H₂O, followed by EtOAc to obtain 11a as a tan solid (25 mg, 4% yield): mp 185 °C (dec); ¹H NMR (DMSO-d₆) δ 2.05 (quint, J = 7.3 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 3.03 (t, J = 7.5 Hz, 2H), 3.81 (s, 2H), 7.04 (s, 2H), 7.26–7.36 (m, 5H); ¹³C NMR (DMSO-d₆) 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. for C₁₅H₁₆N₆O₁S₂·0.02H₂O·0.1EtOAc·0.06NaCl: C, 49.57; H, 4.55; N, 22.52; S, 17.19. Found: C, 49.81; H, 4.46; N, 22.92; S, 16.79.

5-(5-amino-1,3,4-thiadiazol-2-yl)pentanoic acid (9b)

Methyl adipoyl chloride 8b (25 g, 0.14 mol) and thiosemicarbazide (12.8 g, 0.14 mol) were dissolved in POCl₃ (50 mL) and heated to 85 °C 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 g, 89% yield): ¹H NMR (DMSO-d₆) δ 1.49–1.65 (m, 4H), 2.23 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 7.2 Hz, 2H), 7.00 (br s, 2H), 12.02 (br s, 1H).

5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)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 30 min and the solid was filtered to obtain 10b as a off-white solid (44 g, 92% yield): ¹H NMR (DMSO-d₆) 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), 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).

N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (11b)

Compound 10b (1.2 g, 3.8 mmol) and thiosemicarbazide (0.34 g, 3.8 mmol) were dissolved in POCl₃ (5 mL) and heated to 85 °C for 4 h. The reaction was cooled and the viscous oil was poured onto ice. The solution was basified using NaOH pellets to pH ~5. The precipitate was filtered off to obtain off-white solid. The solid was triturated with boiling water (~100 mL), followed by hot EtOAc to afford 11b as a off-white solid (0.63 g, 45% yield): mp 210 °C (dec); ¹H NMR (DMSO-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, 2H), 7.23–7.35 (m, 5H), 12.67 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 28.1, 28.2, 28.4, 28.9, 41.5, 126.9, 128.4, 129.2, 134.6, 158.0, 158.2, 163.8, 168.2, 169.3. Anal. Calcd. for C₁₆H₁₈N₆O₁S₂·0.4H₂O: C, 50.35; H, 4.96; N, 22.02; S, 16.80. Found: C, 50.67; H, 4.73; N, 21.71; S, 16.40.

Methyl 5-(5-amino-1,3,4-thiadiazol-2-yl)pentanoate (12)

To a solution of carboxylic acid 9b (4.0 g, 20 mmol) in MeOH (100 mL), was added concentrated H₂SO₄ (4 mL). The reaction mixture was stirred at 65 °C for 2 d. MeOH was removed and the product was extracted with saturated NaHCO₃ (100 mL) and DCM (4 × 50 mL). The organic layer was dried over MgSO₄ and concentrated in vacuo to obtain a white residue. The residue was triturated with Et₂O to afford 12 as a white solid (3.0 g, 71% yield): ¹H NMR (DMSO-d₆) δ 1.56–1.62 (m, 4H), 2.34 (t, J = 7.2 Hz, 2H), 2.79 (t, J = 7.2 Hz, 2H), 3.58 (s, 3H), 7.00 (s, 2H).

Methyl 5-(5-(3-phenylpropanamido)-1,3,4-thiadiazol-2-yl)pentanoate (13a)

To compound 12 (1.0 g, 4.6 mmol) in DCM (20 mL) was added NEt₃ (1.4 mL, 10 mmol). This was followed by addition of 3-phenylpropanoyl 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 MgSO₄, concentrated in vacuo to obtain off-white solid. The solid was triturated with Et₂O to obtain 13a as a white solid (1.5 g, 95% yield): ¹H NMR (DMSO-d₆) 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, 2H), 2.92–2.99 (m, 4H), 3.58 (s, 3H), 7.17–7.30 (m, 5H), 12.42 (s, 1H).

Methyl 5-(5-benzamido-1,3,4-thiadiazol-2-yl)pentanoate (13b)

Compound 13b was prepared from compound 12 as described for the preparation of 13a except benzoyl chloride was used in place of 3-phenylpropanoyl chloride. The crude material was purified by trituration with EtOAc: white solid (48% yield); ¹H NMR (DMSO-d₆) δ 1.57–1.64 (m, 2H), 1.70–1.78 (m, 2H), 2.37 (t, J = 7.3 Hz, 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).

Methyl 5-(5-(2-cyclohexylacetamido)-1,3,4-thiadiazol-2-yl)pentanoate (13c)

Compound 13c was prepared from compound 12 as described for the preparation of 13a except 2-cyclohexylacetyl chloride was used in place of 3-phenylpropanoyl chloride: white solid (59% yield); ¹H NMR (CDCl₃) 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), 3.02 (t, J = 7.5 Hz, 2H), 3.65 (s, 3H), 12.45 (br s, 1H).

Methyl 5-(5-(2-p-tolylacetamido)-1,3,4-thiadiazol-2-yl)pentanoate (13d)

Compound 13d was prepared from compound 12 as described for the preparation of 13a except 2-p-tolylacetyl chloride was used in place of 3-phenylpropanoyl chloride: white solid (86% yield); ¹H NMR (DMSO-d₆) δ 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 5-(5-(2-(4-fluorophenyl)acetamido)-1,3,4-thiadiazol-2-yl)pentanoate (13e)

Compound 13e was prepared from compound 12 as described for the preparation of 13a except 2-(4- fluorophenyl)acetyl chloride was used in place of 3-phenylpropanoyl chloride: white solid (84% yield); ¹H NMR (CDCl₃) 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 = 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 5-(5-(2-phenylpropanamido)-1,3,4-thiadiazol-2-yl)pentanoate (13f)

Compound 13f was prepared from compound 12 as described for the preparation of 13a except 2-phenylpropanoyl chloride was used in place of 3-phenylpropanoyl chloride: white solid (74% yield); ¹H NMR (CDCl₃) 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), 3.05 (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).

5-(5-(3-phenylpropanamido)-1,3,4-thiadiazol-2-yl)pentanoic acid (14a)

To 13a (1.5 g, 4.4 mmol) in MeOH (70 mL) and H₂O (30 mL) was added LiOH (11 mmol). The reaction mixture was stirred at 40 °C for 24 h. MeOH was removed and solution was diluted with ~20 mL H₂O. The reaction was acidified to pH ~ 2. The precipitate was collected by vacuum filtration to obtain 14a as a white solid (1.0 g, 69% yield): ¹H NMR (DMSO-d₆) δ 1.51–1.58 (t, J = 7.5 Hz, 2H), 1.66–1.74 (t, 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).

5-(5-benzamido-1,3,4-thiadiazol-2-yl)pentanoic acid (14b)

Compound 14b was prepared as described for the preparation of 14a except methyl 5-(5-benzamido-1,3,4-thiadiazol-2-yl)pentanoate 13b was used in place of 13a: off-white solid (83% yield); ¹H NMR (DMSO-d₆) δ 1.58 (m, 2H), 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 = 7.0 Hz, 1H), 8.09 (d, J = 7.1 Hz, 2H), 12.18 (br s, 1H), 12.93 (br s, 1H).

5-(5-(2-cyclohexylacetamido)-1,3,4-thiadiazol-2-yl)pentanoic acid (14c)

Compound 14c was prepared as described for the preparation of 14a except methyl 5-(5-(2-cyclohexylacetamido)-1,3,4-thiadiazol-2-yl)pentanoate 13c was used in place of 13a: white solid (79% yield); ¹H NMR (DMSO-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).

5-(5-(2-p-tolylacetamido)-1,3,4-thiadiazol-2-yl)pentanoic acid (14d)

Compound 14d was prepared as described for the preparation of 14a except methyl 5-(5-(2-p-tolylacetamido)-1,3,4-thiadiazol-2-yl)pentanoate 13d was used in place of 13a: white solid (71% yield); ¹H NMR (DMSO-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).

5-(5-(2-(4-fluorophenyl)acetamido)-1,3,4-thiadiazol-2-yl)pentanoic acid (14e)

Compound 14e was prepared as described for the preparation of 14a except methyl 5-(5-(2-(4-fluorophenyl)acetamido)-1,3,4-thiadiazol-2-yl)pentanoate 13e was used in place of 13a: white solid (63% yield); ¹H NMR (DMSO-d₆) δ 1.54 (m, 2H), 1.69 (m, 2H), 2.24 (t, J = 7.3 Hz, 2H), 2.97 (t, J = 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), 12.66 (br s, 1H).

5-(5-(2-phenylpropanamido)-1,3,4-thiadiazol-2-yl)pentanoic acid (14f)

Compound 14f was prepared as described for the preparation of 14a except methyl 5-(5-(2-phenylpropanamido)-1,3,4-thiadiazol-2-yl)pentanoate 13f was used in place of 13a: white solid (91% yield); ¹H NMR DMSO-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).

N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)-3-phenylpropanamide (15a)

Compound 15a was prepared as described for the preparation of 11b except 5-(5-(3-phenylpropanamido)-1,3,4-thiadiazol-2-yl)pentanoic acid 14a was used in place of 10b. The crude material was purified by trituration with mildly basic water (pH ~ 10, 10mL): off-white solid (33% yield); mp 212 °C (dec); ¹H NMR (DMSO-d₆) δ 1.67–1.66 (m, 4H), 2.75–2.84 (m, 4H), 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 s, 1H); ¹³C NMR (DMSO-d₆) δ 28.2, 28.3, 28.4, 29.0, 30.3, 36.5, 126.1, 128.3, 128.4, 140.6, 158.0, 158.1, 163.4, 168.2, 170.6. Anal. Calcd. for C₁₇H₂₀N₆O₂S·0.7H₂O·0.03NaCl: C, 50.68; H, 5.35; N, 20.68; S, 15.92; Found: C, 51.08; H, 4.95; N, 20.44; S, 15.59.

N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)benzamide (15b)

Compound 15b was prepared as described for the preparation of 11b except 5-(5-benzamido-1,3,4-thiadiazol-2-yl)pentanoic acid 14b was used in place of 10b. The crude material was purified by trituration with mildly basic water (pH ~ 10, 2 × 10mL): tan solid (38% yield); mp 215 °C (dec); ¹H NMR (DMSO-d₆) δ 1.68–1.82 (m, 4H), 2.85 (t, J = 7.2 Hz, 2H), 3.04 (t, J = 7.2 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), 12.83 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 28.3, 28.3, 28.5, 29.0, 128.4, 128.6, 131.8, 132.9, 158.0, 159.4, 164.0, 165.3, 168.2. Anal. Calcd. for C₁₅H₁₆N₆O₂S·0.19EtOAc·0.2NaCl: C, 48.67; H, 4.54; N, 21.61; S,16.49; Found: C, 49.06; H, 4.52; N, 22.00; S, 16.45.

N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-cyclohexylacetamide (15c)

Compound 15c was prepared as described for the preparation of 11b except 5-(5-(2-cyclohexylacetamido)-1,3,4-thiadiazol-2-yl)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), followed by trituration with EtOAc: off-white solid (18% yield); mp 218 °C (dec); ¹H NMR (DMSO-d₆) 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, 2H), 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); ¹³C NMR (DMSO-d₆) 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, 170.7. Anal. Calcd. for C₁₆H₂₄N₆O₁S₂·0.53EtOAc: C, 50.94; H, 6.66; N, 19.67; S, 15.01; Found: C, 51.35; H, 6.25; N, 20.03; S, 14.90.

N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-p-tolylacetamide (15d)

Compound 15d was prepared as described for the preparation of 11b except 5-(5-(2-p-tolylacetamido)-1,3,4-thiadiazol-2-yl)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 MeOH: off-white solid (57% yield); mp 225 °C (dec); ¹H NMR (DMSO-d₆) δ 1.64–1.75 (m, 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), 7.12 (d, J = 8.1 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 12.63 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 20.7, 28.2, 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 C₁₇H₂₀N₆O₁S₂·0.35MeOH·0.1H₂O: C, 51.90; H, 5.42; N, 20.93; S, 15.97; Found: C, 52.23; H, 5.09; N, 20.57; S, 16.20.

N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-(4-fluorophenyl)acetamide (15e)

Compound 15e was prepared as described for the preparation of 11b except 5-(5-(2-(4-fluorophenyl)acetamido)-1,3,4-thiadiazol-2-yl)pentanoic acid 14e was used in place of 10b. The crude material was purified by trituration with mildly basic water (pH ~ 9, 2 × 30 mL), followed by trituration with EtOAc: tan solid (56%); mp 203 °C (dec); ¹H NMR (DMSO-d₆) 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), 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 C₁₆H₁₇N₆O₁FS₂·0.2EtOAc·0.09H₂O·0.4NaCl: C, 46.38; H, 4.35; N, 19.32; S, 14.74; Found: C, 46.78; H, 4.31; N, 18.92; S, 14.36.

N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-phenylpropanamide (15f)

Compound 15f was prepared as described for the preparation of 11b except 5-(5-(2-phenylpropanamido)-1,3,4-thiadiazol-2-yl)pentanoic acid 14f was used in place of 10b. The crude material was purified by trituration with boiling water (~ 50 mL), followed by trituration with MeOH: tan solid (40%); mp 213 °C (dec); ¹H NMR (DMSO-d₆) δ 1.43 (d, J = 8 Hz, 3H), 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), 7.01 (s, 2H), 7.22–7.26 (m, 1H), 7.30–7.37 (m, 4H), 12.56 (br s, 1H); ¹³C NMR (DMSO-d₆) 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 C₁₇H₂₀N₆O₁S₂·0.5H₂O·0.3MeOH: C, 51.04; H, 5.50; N, 20.64; S, 15.75; Found: C, 50.86; H, 5.23; N, 20.49; S, 15.34.

Methyl 7-chloro-6-oxoheptanoate (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 layer was washed with 50 mL of satd NaHCO₃ and 50 mL brine. The organic layer was concentrated and chromatographed in 20% EtOAc/hex with the desired product R_f = 0.4 (20% EtOAc/hex, sulfuric acid charring) to afford 16 (2.9g, 98%): ¹H NMR (CDCl₃) δ 1.66 (m, 4H), 2.35 (m, 2H), 2.67 (m, 2H), 3.67 (s, 3H), 4.08 (s, 2H).

Methyl 5-(2-aminothiazol-4-yl)pentanoate (17)

The α-chloroketone 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 NaHCO₃ (20 mL). The DCM layer was dried and concentrated to afford a white solid that was triturated with Et₂O to afford 2.5 g of the desired aminothiazole 17 (81%): mp 123–128 °C; ¹H NMR (CDCl₃) δ, 1.67 (m, 4H), 2.33 (m, 2H), 2.54 (m, 2H), 3.66 (s, 3H), 4.95 (br s, 2H), 6.10 (s, 1H).

Methyl 5-(2-(2-phenylacetamido)thiazol-4-yl)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 NaHCO₃ (20 mL). The DCM layer was dried and concentrated, then chromotographed on silica gel (1/1 hexanes/EtOAc) to afford 2.5 g (81%) of the desired methyl ester 18: ¹H NMR (CDCl₃) δ 1.64 (m, 4H), 2.31 (m, 2H), 2.61 (m, 2H), 3.65 (s, 3H), 3.81 (s, 2H), 6.52 (s, 1H), 7.39–7.27 (m, 5H), 8.95 (br s, 1H).

5-(2-(2-phenylacetamido)thiazol-4-yl)pentanoic acid (19)

The methyl ester 18 (2.0 g) was 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 with water and then triturated with ether to afford 19 (1.75 g, 92% yield): ¹H NMR (DMSO-d₆) δ 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).

N-(4-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)thiazol-2-yl)-2-phenylacetamide (20)

Compound 20 was prepared as described for the preparation of 11b except 5-(2-(2-phenylacetamido)thiazol-4-yl)pentanoic acid 19 was used in place of 10b: off-white solid (29% yield); mp 192–197 °C; ¹H NMR (DMSO-d₆) δ 1.63–1.65 (m, 4H), 2.61 (t, J = 6.8 Hz, 2H), 2.80 (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); ¹³C NMR (DMSO-d₆) 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, 158.2, 168.1, 169.0. Anal. Calcd. for C₁₇H₁₉N₅O₁S₂·0.5H₂O·0.02EtOAc: C, 53.39; H, 5.29; N, 18.22; S, 16.69. Found: C, 53.75; H, 5.11; N, 17.88; S, 16.31.

Ethyl 7-oxoheptanoate (22)

Compound 22 was prepared from acid chloride 21 as previously described.²² The aldehyde 22 was used without further purification: red oil (95% yield); ¹H NMR (CDCl₃) 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 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 6-bromo-7-oxoheptanoate (23)

Compound 23 was prepared from aldehyde 22 as was previously described.²² The bromide 23 was used without further purification: orange oil (62% yield); ¹H NMR (CDCl₃) δ 1.24 (t, J = 7.2 Hz, 3H) 1.62–1.75 (m, 4H), 2.28 (t, J = 7.5 Hz, 1H), 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 5-(2-aminothiazol-5-yl)pentanoate (24)

Compound 24 was prepared from α-bromoaldehyde 23 as was previously described.²² The crude material was purified by column chromatography (99.9% EtOAc, 0.1% NH₄OH): brown oil (31% yield); ₁H NMR (CDCl₃) δ 1.22 (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 5-(2-(2-phenylacetamido)thiazol-5-yl)pentanoate (25)

Compound 25 was prepared as described for the preparation of 18 except ethyl 5-(2-aminothiazol-5-yl)pentanoate 24 was used in place of 17: white powder (70% yield); ¹H NMR (CDCl₃) δ 1.22 (t, J = 7.1 Hz, 3H) 1.63–1.67 (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).

5-(2-(2-phenylacetamido)thiazol-5-yl)pentanoic acid (26)

Compound 26 was prepared as described for the preparation of 19 except ethyl 5-(2-(2-phenylacetamido)thiazol-5-yl)pentanoate 25 was used in place of 18: white powder (91% yield); ¹H NMR (DMSO-d₆) δ 1.50–1.56 (m, 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).

N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)thiazol-2-yl)-2-phenylacetamide (27)

Compound 27 was prepared as described for the preparation of 11b except 5-(2-(2-phenylacetamido)thiazol-5-yl)pentanoic acid 26 was used in place of 10b: off-white solid (55% yield); ¹H NMR (DMSO-d₆) δ 1.63–1.65 (m, 4H), 2.61 (t, J = 6.7 Hz, 2H), 2.80 (t, J = 6.8 Hz, 2H), 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, 1H); ¹³C NMR (DMSO-d₆) δ 27.8, 28.5, 29.1, 30.4, 41.7, 107.4, 126.8, 128.4, 129.2, 135.0, 151.0, 157.3, 158.2, 168.1, 169.0. Anal. Calcd. for C₁₇H₁₉N₅O₁S₂·0.01H₂O·0.27MeOH: C, 54.25; H, 5.30; N, 18.32; S, 16.77. Found: C, 54.49; H, 5.05; N, 17.97; S, 16.38.

5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)-N-(2-(pyrrolidin-1-yl)ethyl)pentanamide (29a)

To carboxylic acid 10b (2.5 g, 7.9 mmol) dissolved in DMF was added DIEA (5.5 mL, 32 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 2-(pyrrolidin-1-yl)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 NaHCO₃ (50 mL). The organic layer was dried over MgSO₄ and concentrated to obtain off-white solid. The solid was triturated with EtOAc to obtain 29a as an off-white solid (2.1 g, 65%): mp 152 °C (dec); ¹H NMR (DMSO-d₆) δ 1.52 (quint, J = 3 Hz, 2H), 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), 3.14 (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, 1H); ¹³C NMR (DMSO-d₆) δ 23.0, 24.6, 28.6, 28.7, 34.5, 37.6, 41.6, 53.6, 54.9, 126.9, 128.4, 129.3, 134.7, 158.2, 164.0, 169.3, 171.7. Anal. Calcd. for C₂₁H₂₉N₅O₂ S₁·0.15H₂O·0.2NaCl: C, 58.67; H, 6.87; N, 16.29; S, 7.46. Found: C, 58.28; H, 6.74; N, 16.26; S, 7.30.

N-methyl-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)-N-(2-(pyrrolidin-1-yl)ethyl)pentanamide (29b)

Amide 29b was prepared from 10b as described for the preparation of 29a except N-methyl-2-(pyrrolidin-1-yl)ethanamine 28b was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a. The crude material was purified using column chromatography (5% MeOH, 94% DCM, 1% NH₄OH) to obtain 28a as an orange oil (50% yield): ¹H NMR (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, 2H), 7.25–7.33 (m, 5H). Anal. Calcd. for C₂₂H₃₁N₅O₂S₁·0.3H₂O·0.2NaCl: C, 58.55; H, 7.28; N, 15.24; S, 6.98. Found: C, 58.17; H, 7.11; N, 15.31; S, 7.12.

N-(2-(1-methylpyrrolidin-2-yl)ethyl)-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanamide (29c)

Amide 29c was prepared from 10b as described for the preparation of 29a except 2-(1-methylpyrrolidin-2-yl)ethanamine 28c was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a: tan solid (30% yield); mp 121–131 °C; ¹H NMR (DMSO-d₆) δ 1.25–1.37 (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, 1H); ¹³C NMR (DMSO-d₆) 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, 126.9, 128.5, 129.3, 134.7, 158.2, 164.0, 169.3, 171.5. Anal. Calcd. for C₂₂H₃₁N₅O₂S₁·0.45H₂O: 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.

5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)-N-(2-(piperidin-1-yl)ethyl)pentanamide 29d)

Amide 29d was prepared from 10b as described for the preparation of 29a except 2-(piperidin-1-yl)ethanamine 28d was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a: white solid (39% yield); mp 132 °C (dec); ¹H NMR (DMSO-d₆) δ 1.32–1.35 (m, 2H), 1.41–1.46 (m, 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 H), 7.70 (t, J = 5.6 Hz, 1H), 12.61 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 24.0, 24.6, 25.5, 28.6, 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 C₂₂H₃₁N₅O₂S·0.12H₂O: C, 61.2; H, 7.29; N, 16.22; S, 7.43. Found: C, 60.80; H, 7.09; N, 16.21; S, 7.52.

N-(2-(4-hydroxypiperidin-1-yl)ethyl)-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanamide (29e)

Amide 29e was prepared from 10b as described for the preparation of 29a except 1-(2-aminoethyl)piperidin-4-ol 28e was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a: white solid (15% yield); mp 94–99 °C; ¹H NMR (DMSO-d₆) δ 1.38 (m, 2H), 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 C₂₂H₃₁N₅O₃S·0.25H₂O·0.3NaCl: 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.

5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)-N-(2-(4-phenylpiperidin-1-yl)ethyl)pentanamide (29f)

Amide 29f was prepared from 10b as described for the preparation of 29a except 2-(4-phenylpiperidin-1-yl)ethanamine 28f was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a: off-white solid (18% yield); mp 123–126 °C; ¹H NMR (DMSO-d₆) δ 1.33– 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, 1H), 12.66 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 24.6, 25.2, 28.6, 31.2, 34.9, 36.1, 41.5, 42.1, 53.2, 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 C₂₈H₃₅N₅O₂S·0.22H₂O·0.08NaCl: C, 65.39; H, 6.95; N, 13.62. Found: C, 64.94; H, 6.81; N, 14.10.

N-(2-aminoethyl)-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanamide (29g)

To synthesize 29g, tert-butyl 2-(5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2- yl)pentanamido)ethylcarbamate was first prepared from 10b as described for the preparation of 29a except tert-butyl 2-aminoethylcarbamate was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a. To the solution of 2-(5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2- yl)pentanamido)ethylcarbamate (100 mg, 0.22 mmol) in DCM (2 mL) was added TFA (0.5 mL). The reaction mixture was stirred at room temperature for 2 h. H₂O (20 mL) was added and 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 Et₂O to obtain 95 mg of 29g as a white solid (92% yield): mp 167–172 °C; ¹H NMR (DMSO-d₆) δ 1.54 (quint, J = 7.6 Hz, 2H), 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, 1H); ¹³C NMR (DMSO-d₆) δ 24.2, 28.6, 28.7, 34.8, 36.4, 38.7, 41.5, 126.9, 128.4, 129.2, 134.6, 158.1, 164.0, 169.3, 172.7. Anal. Calcd. for C₁₇H₂₃N₅O₁S₂·TFA·0.2H₂O: C, 47.73; H, 4.93; N, 14.65; S, 6.71. Found: C, 47.38; H, 5.04; N, 14.50; S, 6.91.

(2-(diethylamino)ethyl)-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanamide (29h)

Amide 29h was prepared from 10b as described for the preparation of 29a except N, N-diethylethane- 1,2-diamine 28h was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a: white solid (43% yield); mp 126–132 °C; ¹H NMR (DMSO-d₆) δ 0.91(t, J = 7.1 Hz, 6H), 1.52 (quint, J = 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– 7.35 (m, 5H), 7.69 (t, J = 5.7 Hz, 1H), 12.62 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 11.7, 24.6, 28.6, 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 C₂₁H₃₁N₅O₁S₂: C, 60.40; H, 7.48; N, 16.77; S, 7.68. Found: C, 60.15; H, 7.43; N, 16.66; S, 7.92.

N-tert-butyl-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanamide (29i)

Amide 29i was prepared from 10b as described for the preparation of 29a except 2-methylpropan-2-amine 28i was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a. The crude material was triturated with DCM and Et₂O: white solid (41% yield), mp 158–160 °C; ¹H NMR (DMSO-d₆) δ 1.21 (s, 9H), 1.46–1.53 (m, 2H), 1.59–1.67 (m, 2 H), 2.03 (t, J = 7.2 Hz, 2H), 2.95 (t, J = 7.3 Hz, 2H), 3.79 (s, 2H), 7.24–7.35 (m, 5 H), 7.38 (s, 1H), 12.7 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 24.7, 28.6, 28.6, 35.5, 38.9, 41.6, 49.8, 126.9, 128.5, 129.3, 134.7, 158.2, 164.1, 169.4, 171.4. Anal. Calcd. for C₁₉H₂₆N₄O₂S·0.05H₂O: C, 60.79; H, 7.01; N, 14.92; S, 8.54. Found: C, 60.44; H, 6.95; N, 14.98; S, 8.89.

N-tert-butyl-N-methyl-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanamide (29j)

Amide 29j was prepared from 10b as described for the preparation of 29a except N, 2-dimethylpropan-2-amine 28j was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a. The crude material was purified using column chromatography (3% MeOH, 96% EtOAc, 1% NH₄OH) to obtain 29j as an off-white solid (55% yield): mp 103–109 °C; ¹H NMR (DMSO-d₆) δ 1.30 (s, 9H), 1.50 (quint, J = 7.7 Hz, 2H), 1.68 (quint, J = 7.5 Hz, 2H), 2.28 (t, J = 7.2 Hz, 2H), 2.81 (s, 3H), 2.96 (t, J = 7.3 Hz, 2H), 3.79 (s, 2H), 7.25–7.33 (m, 5 H), 12.67 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 24.3, 27.9, 28.8, 31.6, 35.2, 38.3, 41.6, 55.8, 126.9, 128.5, 129.3, 134.7, 158.2, 164.2, 169.3, 172.3. Anal. Calcd. for C₂₀H₂₈N₄O₂S·0.55 H₂O: C, 60.29; H, 7.36; N, 14.06; S, 8.05. Found: C, 59.91; H, 7.04; N, 13.94; S, 7.80.

N-benzyl-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanamide (29k)

Amide 29k was prepared from 10b as described for the preparation of 29a except benzylamine 28k was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a: white solid (72% yield), mp 186–188 °C; ¹H NMR (DMSO-d₆) δ 1.54–1.61 (m, 2H), 1.63–1.71 (m, 2H), 2.17 (t, J = 7.2 Hz, 2H), 2.96 (t, J = 7.3 Hz, 2H), 3.80 (s, 2H), 4.25 (d, J = 6.1 Hz, 2H), 7.18–7.35 (m, 10H), 8.33 (t, J = 5.8 Hz, 1H), 12.65 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 24.7, 28.6, 28.7, 34.9, 41.6, 42.0, 126.7, 126.9, 127.2, 128.3, 128.5, 129.3, 134.7, 139.7, 158.3, 164.0, 169.4, 171.8. Anal. Calcd. for C₂₂H₂₄N₄O₂S·0.16H₂O: C, 64.23; H, 5.96; N, 13.62; S, 7.79. Found: C, 63.85; H, 5.85; N, 13.52; S, 8.00.

N-(4-(dimethylamino)benzyl)-5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanamide (29l)

Amide 29l was prepared from 10b as described for the preparation of 29a except 4-(aminomethyl)-N,N-dimethylaniline 28l was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a: white solid (32% yield), mp 206 °C (dec); ¹H NMR (DMSO-d₆) δ 1.55–1.67 (m, 4H), 2.13 (t, J = 7.0 Hz, 2H), 2.82 (s, 6H), 2.96 (t, J = 7.3 Hz, 2H), 3.80 (s, 2H), 4.11 (d, J = 5.6 Hz, 2H), 6.65 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 7.25–7.34 (m, 5H), 8.17 (br s, 1H), 12.67 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 24.7, 28.6, 28.7, 34.8, 40.30, 41.55, 41.57, 112.4, 126.9, 127.1, 128.1, 128.5, 129.3, 134.7, 149.5, 158.2, 164.0, 169.3, 171.5. Anal. Calcd. for C₂₄H₂₉N₅O₂S·0.01H₂O: C, 63.81; H, 6.47; N, 15.50; S, 7.10. Found: C, 63.41; H, 6.42; N, 15.50; S, 6.95.

5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)-N-(4-(trifluoromethyl)benzyl)pentanamide (29m)

Amide 29m was prepared from 10b as described for the preparation of 29a except (4-(trifluoromethyl)phenyl)methanamine 28m was used in place of 2-(pyrrolidin-1-yl)ethanamine 28a: white solid (51% yield), mp 214–218 °C; ¹H NMR (DMSO-d₆) δ 1.59 (m, 2H), 1.66 (m, 2H), 2.19 (t, J = 7.2 Hz, 2H), 2.97 (t, J = 7.3 Hz, 2H), 3.79 (s, 2H), 4.33 (d, J = 6.1 Hz, 2H), 7.25–7.33 (m, 5 H), 7.44 (d, J = 7.8 Hz, 2H), 7.67 (d, J = 8.1 Hz, 2H), 8.46 (t, J = 5.9 Hz, 2H), 12.67 (br s, 1H); ¹³C NMR (DMSO-d₆) δ 24.6, 28.6, 28.7, 34.8, 41.6, 41.6, 124.4 (q, J = 272 Hz), 125.2 (q, J = 3.7 Hz), 126.9, 127.4 (q, J = 32 Hz), 127.8, 128.5, 129.3, 134.7, 144.7 (q, J = 1.5 Hz), 158.2, 164.0, 169.4, 172.0. Anal. Calcd. for C₂₃H₂₃N₄O₂F₃S·0.2H₂O: C, 57.54; H, 4.91; N, 11.67; S, 6.68. Found: C, 57.21; H, 4.82; N, 11.67; S, 7.02.

GLS assay

A 96-well microplate-based glutaminase assay was used to determine the activity of various glutaminase inhibitors. The hKGAd1 construct was prepared from the hKGA cDNA26 by deleting the sequence encoded in exon 1 and cloning into pET15b as described for the rat KGAd1 construct.²⁷ Purified human kidney glutaminase (hKGA^124–669; 250 nM) was used as the source of enzyme and radiolabeled glutamine (L-[³H]-glutamine, American Radiolabeled Chemicals, Saint Louis, MO; 2 mM and at a specific activity of 0.91 OCi/Omol) was used as the substrate. The assay was conducted in the presence and absence of inhibitors, at room temperature (45 min incubation), in 45 mM phosphate buffer (pH 8.2). At the end of the reaction period, the assay was terminated upon the addition of 20 mM imidazole buffer (pH 7). 96-well spin columns packed with strong anion ion-exchange resin (Bio-Rad, Hercules, CA) were used to separate the substrate and the reaction product. Unreacted [³H]-glutamine was removed by washing with imidazole buffer. [³H]-Glutamate, the reaction product, was then eluted with 0.1 M HCl and analyzed for radioactivity using Perkin Elmer’s TopCount instrument in conjunction with their 96-well LumaPlates (Waltham, MA).

GLS2 assay

Mouse liver glutaminase assay (mLGA) was carried out using a three enzyme assay system. In the first reaction, glutamine was hydrolyzed to glutamate and ammonia by mouse liver glutaminase.¹⁴ In the second reaction, glutamate was oxidized by glutamate oxidase (Sigma, St. Louis, MO) to α-ketoglutarate, ammonia and hydrogen peroxide. Finally, in the third reaction, hydrogen peroxide was complexed with fluorogenic Amplex red (colorless; Life Technologies, Grand Island, NY) in the presence of horse radish peroxidase (HRP; Worthington Biochemical Corp., Lakewood, NJ) to produce highly fluorescent resorufin (colored product; ex 530, em 590). Enzyme assays were conducted at room temperature in 45 mM phosphate buffer (pH 8.2) containing 0.04 U/mL glutamate oxidase, 0.125 U/mL HRP, 50 OM Amplex UltraRed and glutaminase. The half maximal inhibitory constant (IC₅₀) of various compounds were ascertained in the presence of 1 mM glutamine. In order to rule out assay method dependent variability, we tested compound 6 in fluorescence-based GLS assay and obtained IC₅₀ value of 5.5 ± 0.9 μM.

Determination of aqueous solubility using conventional shake-flask method

The solubility was determined using modified shake-flask procedure.²³ The solid was added to PBS buffer (pH = 7.4) until saturation occurred. To ensure equilibrium is achieved the mixture was shaken for at least 72 h at 37 °C. At the end of the incubation period, the saturated solution was filtered using a syringe filter (0.45 μm PTFE). The filtrate was diluted appropriately and concentration of compound was determined by LC/MS using a calibration curve of DMSO stock solution.

Metabolic stability studies in mouse microsomes

Compounds 6, 11b and 29a were evaluated for phase I and phase II metabolic stability using mouse liver microsomes. For phase I metabolism, the reaction was carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, 0.4 U/mL glucose-6-phosphate dehydrogenase, 50 OM sodium citrate). Reactions in duplicate were initiated by addition of the liver microsomes to the incubation mixture (compound final concentration was 5 OM; 0.5 mg/mL microsomes). For phase II glucuronidation reaction, the compound was added to TRIS-HCl buffer (50 mM, pH 7.5) with microsomes (0.5 mg/mL), along with MgCl₂ (8 mM), and alamethicin (25 Og/mL) and pre-incubated at 37 oC. The reaction was initiated (in duplicate) with UDPGA (2 mM; final concentration). Controls in the absence cofactors were carried out to determine the specific cofactor-free degradation. At predetermined times up to 1 hour, aliquots of the mixture were removed and the reaction quenched by addition of two times the volume of ice cold acetonitrile spiked with the internal standard. Compound disappearance was monitored over time using a liquid chromatography and tandem mass spectrometry (LC/MS/MS) method.

Cell proliferation assay

P493 cells were cultured in RPMI in aerobic conditions in the presence of various concentrations of compound 6. Live cells were counted manually in a hemacytometer using trypan blue dye exclusion method.²⁸

Animal Studies

The animal studies were performed according to the protocols approved by the Animal Care and Use Committee at The Johns Hopkins University. To generate tumorigenesis study in xenograft model, 2.0 × 10⁷ P493 human lymphoma B cells were injected subcutaneously into male SCID mice (National Cancer Institute) as previously described.²⁸ Intraperitoneal (i.p.) injection of compound 6 (200 Og, ~12.5 mg/kg) was performed every other day starting from day 10. Tumor volumes were measured on days 10, 13, 16, and 19. Control animals were treated with i.p. injection of vehicle (2% [vol/vol] DMSO in PBS). The tumor volumes were measured using digital calipers and were calculated using the following formula: [length (mm) × width (mm) × width (mm) ×0.52].

ABBREVIATIONS USED

GLS

kidney-type glutaminase

GLS2

liver glutaminase

BPTES

bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide

UDGPA

UDP-glucuronic acid

HATU

2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate