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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2012 October 13.
Published in final edited form as:
PMCID: PMC3196593
NIHMSID: NIHMS323783

The development of N-α-(2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide (o-F-amidine) and N α-(2-carboxyl)benzoyl-N5-(2-Chloro-1-iminoethyl)-L-ornithine amide (o-Cl-amidine) as second generation Protein Arginine Deiminase (PAD) inhibitors.

Abstract

Protein Arginine Deiminase (PAD) activity is upregulated in a number of human diseases, including rheumatoid arthritis, ulcerative colitis, and cancer. These enzymes, there are five in humans (PADs 1-4 and 6) regulate gene transcription, cellular differentiation, and the innate immune response. Building on our successful generation F- and Cl-amidine, which irreversibly inhibit all of the PADs, a structure activity relationship was performed to develop second generation compounds with improved potency and selectivity. Incorporation of a carboxylate ortho to the backbone amide resulted in the identification of N-α-(2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide (o-F-amidine) and -(2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (o-Cl-amidine), as PAD inactivators with improved potency (up to 65-fold) and selectivity (up to 25-fold). Relative to F- and Cl-amidine, the compounds also show enhanced potency in cellulo. As such, these compounds will be versatile chemical probes of PAD function.

It has long been recognized that posttranslational modifications (PTMs) play critical roles in maintaining the homeostasis of biological systems. While many PTMs, and their putative roles in disease etiology, are well documented (e.g., protein phosphorylation), most others are less well understood. One example of the latter is protein deimination (Figure 1A).1 This PTM is generated by the hydrolytic removal of ammonia from the guanidinium of a substrate arginine residue to generate a ureido group.1, 2 Because this modification results in the formation of citrulline, this process is also termed citrullination. The enzymes responsible for generating this PTM are known as the Protein Arginine Deiminases (PADs), and in humans, and other mammals, there are five isozymes (i.e., PADs 1, 2, 3, 4, and 6) that possess a high degree of inter-isozyme sequence homology (~50 %).1, 3 Given that these isozymes possess similar, but not identical, substrate specificities,4, 5 it is perhaps not surprising that the major distinguishing feature of individual family members is their tissue distribution patterns.3 For example, whereas PAD2 is near universally expressed in all tissue and cell types, the expression patterns of the remaining PADs are much more restricted, with PADs 1, 3, 4, and 6 being most predominantly expressed in the skin, hair follicles, immune cells, and oocytes, respectively.3

Figure 1
Protein arginine deiminase reaction and inhibitors. (A) The guanidinium group of an arginine residue is converted into a ureido group to form citrulline. (B) Structures of Cl- and F-amidine.

Although one or more of these enzymes are expressed in virtually all cell types, our understanding of their physiological functions, especially with regard to PADs, 1, 2, 3 and 6, is limited. However, it is known that these enzymes play incompletely defined roles in a diverse number of processes, including: the cornification of the skin (PADs 1, 2, and 3), hair follicle formation (PAD3), nerve myelination (PAD2), and fertility (PAD6).3, 6-10 Given the early links suggesting that dysregulated PAD4 activity contributes to the onset and progression of rheumatoid arthritis (RA; see below and reference 1 for a review), our understanding of the physiological roles of PAD4, while still limited, is more advanced. For example, a fraction of PAD4 is localized to the nucleus where it has been shown to modify the unstructured tails of histones H3 and H4, and this modification has been associated with the decreased transcription of genes under the control of a number of transcription factors, including the estrogen receptor, thyroid receptor, and p53.11-14 More recently, PAD4 was shown to deiminate ELK1, and this PTM was associated with increased transcription of the proto-oncogene c-Fos.15

In addition to modulating gene transcription, PAD4 activity has been shown to affect a variety of processes, including apoptosis – inhibition and overexpression of PAD4 induces apoptosis13, 14, 16 – and differentiation – PAD4 inhibition triggers the differentiation of HL60 cells into HL60 granulocytes and monocytes.17 Emerging evidence also suggests that PAD4 may play a key role in regulating at least a subset of the innate immune response. For example, PAD4 has been shown to deiminate a number of chemokines (e.g., CXCL 5, 8, 10, 11, and 12), and this modification reduces their ability to trigger chemotaxis and cell signaling in vitro.18-20 Additionally, PAD4 activity appears to be critical for the formation of Neutrophil Extracellular Traps (NETs), as Cl-amidine (Figure 1B), a pan-PAD inhibitor developed by our lab,21 prevents NET formation;22, 23 neutophils extravate their chromatin to form net-like structures in response to a number of signaling molecules of both bacterial (e.g., LPS and fMLP)22-24 and human origin (e.g., CXCL2 and CXCL8).25

Dysregulated PAD expression and activity has been associated with a number of human diseases, including, RA, Ulcerative Colitis, Crohn’s Disease, Glaucoma, Multiple Sclerosis, Alzheimer’s disease, and even Cancer (for a review see 1). Although the association between dysregulated PAD activity and human disease seems clear, especially with our recent demonstration that Cl-amidine decreases disease severity in the collagen induced arthritis (CIA) model of RA and the dextran sodium sulphate (DSS) model of Ulcerative Colitis,26, 27 what is not known are the specific identities of the isozymes involved in the onset and progression of these pathologies. From the available evidence, the most likely candidates appear to be PADs 2 and 4, as these enzymes are expressed by immune cells (the diseases listed above all have an autoimmune component) and both are overexpressed in these diseases.1, 3, 28, 29 With respect to RA, the evidence linking dysregulated PAD4 activity to the onset and progression of this disease are particularly strong. For example, patients with RA produce autoantiboides targeting both citrullinated proteins and PAD4 itself, and these autoantibodies first appear in the preclinical phase of the disease.30-33 Additionally, mutations in the gene encoding PAD4 confer an increased risk of developing RA, at least in Asian populations.34, 35 Finally, PAD4 and citrullinated proteins are present in the inflamed joints of RA patients.28, 36 However, the fact that PAD2 is also overexpressed in these tissues28 complicates the seemingly simple picture that dysregulated PAD4 activity is the only PAD that contributes to the etiology of RA. Thus, it is unclear whether the inhibition of single or multiple PADs is required to treat these diseases. One way to address this issue is the development of isozyme-specific PAD inhibitors that can be used to discern the relative contributions of individual PADs to human disease. To begin to develop such compounds, and also identify compounds with enhanced potency and bioavailability, we performed Structure Activity Relationships (SAR) on our previously described PAD inhibitors F- and Cl-amidine (Figure 1B). Herein we describe the design, synthesis, and testing of a series of new PAD inhibitors with enhanced potency, selectivity, and bioavailability. In particular, we describe the development of o-F-amidine and o-Cl-amidine as two of the most potent and partially selective PAD inhibitors described to date.

Results and Discussion

Design, synthesis, and characterization of X-amidine side chain analogs

The structure of F-amidine, which is used as a baseline for comparison, can be divided into three main portions: the warhead, the side chain, and the backbone (Figure 1B). To better understand the relative contribution of the side chain to inhibitor potency, we initially synthesized 2-fluoro-N-propylacetamidine (4) (Figure 2), which is essentially a propylamine modified warhead, to mimic the three-carbon side chain portion of F-amidine. The compound was synthesized by reaction of propylamine with 2-fluorothioacetimidate, purified, and IC50 values obtained for PAD4. The results indicate that 4 is a relatively weak PAD4 inhibitor (IC50 > 1 mM; Table 2), a value that is comparable to that obtained for the warhead, i.e. 3, alone. The fact that F-amidine is approximately two orders of magnitude more potent than 4 indicates that the side chain component of F-amidine is not responsible for the relatively high potency of F-amidine.

Figure 2
Structures of side chain analogs (A) and backbone analogs (B).
Table 2
IC50 values of PAD4 inhibitorsa

Given that a tryptophan residue (i.e., W347) lines the active site of PAD4 and interacts with the side chain portion of F-amidine via hydrophobic and Van der Waals interactions (Figure 3), we considered the possibility that the incorporation of an aromatic moiety, in place of the aliphatic side chain, would interact favorably with the W347 indole group. To test this hypothesis, the fluoroacetamidine warhead was installed on two structurally similar aromatic amines, i.e. aniline and benzylamine. However, neither the benzyl (5: IC50 > 1 mM) nor the aniline (6: IC50 > 500 μM) derivatives were potent PAD4 inhibitors. Nevertheless, the fact that the aniline derivative was more potent than the baseline compound, i.e. the propyl derivative, suggested that its incorporation into the ‘full inhibitor’ might enhance inhibitor potency. To examine this possibility, we synthesized and compared the relative potencies of phenylalanine and phenylglycine derivatives in which the warhead is placed meta to the α-carbon. Note that both the phenylalanine and phenylglycine derivatives were synthesized to explore how differences in the connectivity to the backbone affect inhibitor potency. The results of these SAR studies indicated that the phenylalanine derivative, i.e. 7, (IC50 = ~300 μM) was more potent than the phenylglycine derivative (8) (> 2 mM) suggesting that the extra methylene unit between the backbone and warhead is advantageous. Given that these values are significantly less than those obtained with F-amidine, and represent only a slight improvement over the aniline derivative on its own, we considered the possibility that the lack of potency was due to a less than ideal positioning of the warhead about the phenyl ring. To partially test this hypothesis, two para-substituted phenylalanine derivatives, i.e. compounds 9 and 10, were synthesized. The first compound attaches the warhead directly to the phenyl ring, mimicking the structure of the aniline, and the second compound attaches the warhead via a one carbon methylene linker that was expected to project the warhead further into the active site where it might react more favorably with the enzyme. Unfortunately, the IC50 values obtained for compounds 9 and 10 (IC50 ~ 300 μM and > 1 mM, respectively) are similar to those determined for the ortho substituted derivatives, thereby indicating that the replacement of the aliphatic side chain of F-amidine with a phenyl moiety does not lead to inhibitors with enhanced potency.

Figure 3
Crystal structure of PAD4•F-amidine complex. This figure was prepared with UCSF Chimera using the coordinates for the PAD4•F-amidine complex (PDBID: 2DW5).

Design, synthesis, and characterization of X-amidine backbone analogs

Since none of the side chain analogs were more potent than F-amidine, we considered whether changes to the backbone might yield inhibitors with enhanced potency. The backbone component of F-amidine contains two amide bonds that are presumed, although not yet proven, to be important for binding to the enzyme. To determine whether this was indeed the case, we synthesized compound 11 (Figure 2B), which lacks the C-terminal carboxamide moiety. The IC50 value obtained for this compound (IC50 ≥ 500 μM) confirms that both amide groups are critical for the overall potency of F-amidine. We next considered the effect of substituting the benzoyl-carboxamide for a sulfonamide (12). While this substitution retains the H-bond acceptor, and in fact introduces a second one, the spatial orientation is somewhat different – the sulfonamide has a tetrahedral geometry while the carboxamide has a trigonal planar geometry. Although it was impossible to predict exactly how this substitution would affect the potency, the experimental evidence (IC50 > 1 mM) clearly indicates that the effect was deleterious. To determine whether the lack of potency was solely due to the loss of the C-terminal carboxamide, the introduction of the sulphonamide, or a combination of both changes, we next synthesized compound 13, which retains the C-terminal carboxamide, but replaces the benzoyl-carboxamide with a sulfonamide. Interestingly, this compound was also a poor PAD4 inhibitor, which, combined with the results described above for compounds 11 and 12, suggests that hydrogen bonding between R374 and the two backbone carbonyls contributes to the potency of F-amidine in a synergistic fashion.

Given that substitutions altering the two backbone carbonyls, as well as the side chain, yielded inhibitors with decreased potency, we considered that substitutions of the benzoyl group might yield inhibitors with increased potency. In particular, we considered substitutions that would mimic an extension of the peptide chain. Our first attempt replaced the phenyl ring with an imidazole ring (14). This substitution maintains the aromatic character of the phenyl ring while incorporating an NH moiety that might mimic a peptide NH, thus becoming a hydrogen bond donor – an additional hydrogen bond could significantly increase the binding affinity of the inhibitor. The results of inhibition studies with 14 showed a 10-fold decrease in potency relative to F-amidine (IC50 = 230 ± 110 μM). This result was somewhat unexpected given that even in the absence of an additional H-bond, the imidazole moiety was still aromatic and thus should be, at worst, comparable to the phenyl moiety. Upon further consideration, however, we realized that the imidazole was likely protonated under the reaction conditions resulting in a positively charged species. Therefore, we concluded that the decrease in potency may actually arise from a charge-charge repulsion between the protonated imidazole and a positively charged residue on the enzyme.

In order circumvent this potential charge-charge repulsion, we chose to substitute the phenyl ring with a pyrollidine ring (15). This substitution maintains both the aromaticity and the NH bond as a potential H-bond donor, but given the much higher pKa, the pyrollidine remains neutral under the assay conditions. Synthesis and evaluation of this derivative was carried out and the results indicate a potency that is comparable to F-amidine. Although this inhibitor is not more potent than the parent compound, the fact that the IC50 is the same as the benzoylated version suggests two things: i) the NH may contribute little to overall binding; and ii) the loss of potency seen with the imidazole derivative is likely due to a charge-charge repulsion.

Because the results from the imidazole and pyrollidine derivatives suggest that the presence of a positively charged residue in close proximity to the active site, we postulated that the incorporation of a negatively charged moiety on the benzoyl group would increase inhibitor potency. Towards this end, we chose to substitute the phenyl ring of F-amidine with a paranitro-phenol (16). We postulated that the positioning of the nitro group para to the hydroxyl would sufficiently depress the pKa of the hydroxyl to generate an inhibitor that is at least partially deprotonated. This negative charge, should then be positioned to interact with the positively charged residue on the enzyme without being sterically cumbersome. Once this derivative was synthesized and tested, the results showed a small decrease (~2-fold) in potency (IC50 = 12.1 ± 6.4 μM).

Given these promising results, we next designed and synthesized an F-amidine derivative that incorporated a carboxylate ortho to the backbone amide (17). Synthesis of this derivative, which is denoted o-F-amidine, was accomplished by using phthalic anhydride in the place of benzoyl chloride. As predicted, the IC50 of this compound (1.9 ± 0.21 μM) is 10-fold lower than that obtained with the parent compound, F-amidine. Given that the substitution of chloride for fluoride typically increases the potency of X-amidine inhibitors by ~5-fold, we anticipated a similar trend in these derivatives as well. However, once the chloride derivative was synthesized and assayed, we found that the inhibitory potency of o-Cl-amidine (18) (IC50 = 2.2 ± 0.31 μM) was nearly identical to that of o-F-amidine.

Mechanism of Inhibition

Since the parent compounds, i.e. F- and Cl-amidine, are mechanism based inactivators, we next determined whether o-F- and o-Cl-amidine also irreversibly inactivate PADs 1, 2, 3, and 4. For these studies, a pre-formed complex of inactivator and enzyme was generated, then dialyzed for 20 h, at which point the residual activity was measured. As shown in Figure S1, little to no recovery of activity was observed for all of the PADs tested, thereby confirming that, like F- and Cl-amidine, o-F- and o-Cl-amidine are irreversible PAD inactivators. In order to help confirm that PAD inactivation is due to the modification of an active site residue, substrate protection experiments were performed. For these studies, product formation was measured as a function of time at 2 different concentrations of substrate (10 and 2 mM benzoyl-L-arginine ethyl ester (BAEE)) with and without inactivator. The results (Figure S2) indicate that for PADs 1, 2, 3, and 4 the rates of inactivation are slower at higher substrate concentrations. Such a result is most consistent with enzyme inactivation being due to the modification an active site residue, which, based on precedence, is likely the active site cysteine.

Selectivity Studies

To evaluate the selectivity of o-F- and o-Cl-amidine, kinact, KI, and kinact/KI values were determined for all of the PADs (Table 3). Note that for irreversible inhibitors these rate constants provide a more accurate assessment of selectivity than IC50 values. For these experiments, the residual activity was measured as a function of time with increasing concentrations of inactivator (see Figure 4A and 4C for representative data). From these curves, the pseudo first order rate constants of inactivation (i.e., kobs) were determined and plotted against inactivator concentration and fit to equation 4 to provide values for kinact, KI, and kinact/KI (see 4B and 4D for representative data). Interestingly, based on the kinact/KI values, o-Cl-amidine preferentially inactivates PAD1, as the kinact/KI value obtained for this isozyme is 8-, 10-, 3-fold higher than that obtained for PADs 2, 3, and 4, respectively. Similar trends were observed for o-F-amidine, where the kinact/KI values are 24-, 27-, and 6-fold higher for PAD1 than for PADs 2, 3, and 4, respectively. Given that the relative selectivity of o-Cl-amidine for PADs 1 and 4 is relatively modest (~3-fold), this compound is a PAD1 and 4 selective inhibitor, whereas o-F-amidine is primarily a PAD1 selective inhibitor. It is also important to note that o-F-amidine is 60-fold more potent than the parent compound F-amidine.

Figure 4
Inactivation of PAD4 with o-F- and o-Cl-amidine. The residual activity of PAD4 was measured versus time with increasing concentrations of (A) o-F-amidine and (C) o-Cl-amidine. The rates (kobs) were plotted versus concentration of (B) o-F-amidine and (D) ...
Table 3
Inhibition of PAD isozymes by haloacetamidine-based inhibitors.

Structural Studies

To help determine the molecular basis for the enhanced potency of both o-F- and o-Cl-amidine, the structures of these compounds bound to the PAD4•calcium complex were determined and compared to the structure of the PAD4•calcium•F-amidine (Figure 5A and 5B). The structures of these complexes are virtually superimposable (rmsd = 0.222 Å for comparable 573 Cα), and revealed the presence of three potential residues, i.e. Q346, W347, and R374, that could provide favorable hydrogen bonding and/or electrostatic interactions with the ortho carboxylate that would explain the enhanced potency of o-F- and o-Cl-amidine (Figure 5C). While the distance between the indole NH of W347 and the ortho carboxylate is characteristic of a hydrogen bond (i.e., 2.9 Å), the distances between the ortho carboxylate and the carboxamide of Q346 (i.e., 3.9 Å) and the guanidinium group of R374 (i.e., 4.6-5.6 Å) are greater than that typically associated with such an interaction. Note that in the o-F-amidine complex, water-mediated hydrogen bonds between Q347 and the carboxylate of o-F-amidine are observed, because of higher-resolution data. Given that these structures represent the dead end complex, and as such do not represent the initial encounter complex where the effects of the ortho carboxylate on enzyme inactivation are expected to be most profound, we used site directed mutagenesis to identify the residue(s) that is/are most critical for inhibitor recognition. Specifically, Q346, W347, and R374 were individually converted to alanine and IC50 values were determined for the mutant enzymes. To aid our analysis, we also generated the Q346E, W347F, and R374K mutants. The results of these studies (Table 4) indicate that mutation of Q346 to either an alanine or a glutamate had essentially no effect on the IC50 values determined for o-F- and o-Cl-amidine, suggesting that the enhanced potency of these compounds is not due to an interaction between the carboxylate and carboxamide moieties on the inhibitor and Q346, respectively. In contrast, the IC50 values determined for the R374K and R374A mutants are ~2.4-fold and 28-fold higher for o-F-amidine when compared to the values obtained for wild type enzyme. Since similar effects on potency were observed for F-amidine, these results suggest that the interaction between the ortho-carboxylate and the R374 guanidinium is not crucial for the enhanced the potency of these PAD inactivators. Nevertheless, this arginine residue is conserved in both PADs 1 and 4, and the presence of R374 may explain why o-F- and o-Cl-amidine preferentially inactivate these enzymes. Unfortunately, neither of the tryptophan mutants were active (kcat/Km ≤ 0.2 M−1 s−1), thus the importance of the indole NH group to inhibitor potency could not be evaluated directly. Nevertheless, this interaction is likely important when one considers the fact that the introduction of the ortho carboxylate led to enhanced potency among all the isozymes and the fact that this residue is universally conserved among the PAD isozymes.

Figure 5
Overlays of the crystal structure of PAD4 with (A) F-amidine and o-F-amidine bound and (B) Cl-amidine and o-Cl-amidine bound. (C) Stereoview of the crystal structure of PAD4 bound to o-F-amidine.
Table 4
PAD4 IC50 values

Bioavailability of o-F- and o-Cl-amidine

To illustrate the utility of o-F- and o-Cl-amidine as chemical probes of in vivo PAD4 function, we set out to evaluate their activity in cellulo. Initially, the bioavailability of the compounds was determined by monitoring their effects on histone H3 citrullination in HL60 granulocytes; PAD4 is known to deiminate histone H3 in this cell line in response to the calcium ionophore A23187.37, 38 Western blots, using an anti-citrullinated histone H3 antibody, demonstrated that the levels of deiminated histone H3 are decreased upon a 30 min incubation with the inhibitors. Significantly, the improved in vitro potency of o-F- and o-Cl-amidine is paralleled in cellulo, as exemplified by the fact that as little as 1 μM of o-F-amidine decreased the levels of deiminated histone H3 to the same extent as 100 μM of Cl-amidine (Figure 6A).

Figure 6
(A) Levels of citrullinated histone H3 in HL-60 granulocytes after treatment with o-F- and o-Cl-amidine. (B) o-F- and o-Cl-amidine induce the differentiation of HL-60 cells. HL-60 cells were exposed to either o-F-, or o-Cl-amidine over the course of 48 ...

Effect of o-F- and o-Cl-amidine on cell viability

Having demonstrated that the additional carboxylate does not affect the bioavailability of these inhibitors, we next investigated whether these compounds potentiate the cell killing effects of doxorubicin, similarly to the parent compounds F- and Cl-amidine.17 For these experiments, the effect of doxorubicin on HL-60 cell survival was determined in the absence and presence of increasing concentrations of o-F- and o-Cl-amidine using the standard MTT assay. As depicted in Figure 7, addition of o-F- and o-Cl-amidine caused the dose response curves to shift to the left, resulting in an approximate 6-fold decrease in the EC50 for doxorubicin (Table 5). The fact that the magnitude of the effect is similar for both o-F- and o-Cl-amidine is consistent with the fact that these compounds are equipotent PAD4 inhibitors. Relative to 1 μM of Cl-amidine, which reduced the EC50 by ~2-fold, both o-F- and o-Cl-amidine decreased the EC50 by ~6-fold at concentrations as low a 100 nM. In addition to decreasing the EC50 of doxorubicin, both compounds also decrease cell survival to background levels, i.e. no viable cells were detectable. To determine whether the effects imparted by o-F- and o-Cl-amidine on doxorubicin cell cytotoxicity were sub-additive, additive, or synergistic, the additive model was used.39, 40 This model predicts that two drugs are sub-additive when the observed/expected (O/E) ratio is 1.2, additive when the O/E ratio is between 0.8 and 1.2, and synergistic when the O/E ratio is < 0.8. As depicted in Figure 7C and 7D, o-F- and o-Cl-amidine potentiate the cell killing of doxorubicin in HL-60 cells in a synergistic and dose dependent fashion.

Figure 7
Effects of o-F-amidine (A) and o-Cl-amidine (B) in combination with doxorubicin on cell viability of HL-60 cells. Cell viability was measured, using a standard MTT assay, after a 24 h incubation with the inhibitors. Each data point represents an average ...
Table 5
Cytotoxicity of o-F- and o-Cl-amidine in combination with Doxorubicin in HL-60 cells.

o-F- and o-Cl-amidine mediated differentiation of HL60 cells

Given that F- and Cl-amidine can trigger the differentiation of multiple cancer cell lines,17 including the differentiation of HL60 cells into HL60 granulocytes, we evaluated whether o-F- and o-Cl-amidine can also act as differentiating agents. For these experiments, PAD4 expression levels were monitored after the treatment of HL60 cells with o-F- and o-Cl-amidine; PAD4 expression levels are increased upon the differentiation of HL60 cells. Western blots, using an anti-PAD4 antibody, indicate that o-F- and o-Cl-amidine treatment causes a dose and time dependent increase in PAD4 protein expression. For example, at the highest doses of o-F- and o-Cl-amidine tested (i.e., 10 μM), PAD4 expression was apparent after only 6 h of treatment; however, with lower doses (e.g. 1 μM) increased levels of this enzyme were observed only after 12 and 24 h. In total, the results of these experiments demonstrate that these inhibitors differentiate HL-60 cells into HL-60 granulocytes similarly to the parent compounds.

Inflammatory cell apoptosis

We have recently shown that Cl-amidine induces inflammatory cell apoptosis in vitro and in vivo27 and have suggested that this inhibitor may suppress colitis by removing the cells that drive colitis. With the increased potency of o-F-amidine, we thought it would be prudent to test the hypothesis that o-F-amidine has a more potent impact on inflammatory cell apoptosis than Cl-amidine. Results shown in Figure 8 are consistent with this hypothesis. For example, representative scatterplots of Annexin V/Propidium Iodide staining following exposure of TK6 lymphoblastoid cells to o-F-amidine (50 μM) or Cl-amidine (50 μM) for up to 8 hours demonstrate that o-F-amidine increases the number of apoptotic cells to a greater extent than an equivalent concentration of Cl-amidine (Figure 8A). Figure 8B depicts the quantification of apoptosis (4th quadrant). Additionally, o-F-amidine induces PARP cleavage earlier and to a greater extent than the same concentration of Cl-amidine (Figure 8C).

Figure 8
o-F-amidine is more potent at driving apoptosis in inflammatory cells than Cl-amidine. TK6 cells, a human lymphoblastoid cell line, were exposed to either Cl-amidine (50 μg/ml) or o-F-amidine (50 μg/ml) for indicated time periods. (A) ...

Conclusions

In conclusion, SAR led to the development of two new PAD inhibitors, o-F- and o-Cl-amidine, that are significantly (up to 65-fold) more potent than F- and Cl-amidine. The increased potency is due to the carboxylate functionality in the ortho position of the benzoyl ring. Although the inclusion of this group enhanced potency for all of the PADs, the effects are isozyme and warhead dependent. For example, with o-Cl-amidine, the increase in kinact/KI ranged from 3-fold for PADs 1 and 4 to 12-fold for PAD2 whereas for o-F-amidine the kinact/KI values were increased by 11-, 20-, 39-, and 65-fold for PADs 4, 2, 3, and 1, respectively. The observed warhead dependence on kinact/KI likely reflects differences in the ability to position the warhead for nucleophilic attack by the active site cysteine. This result is significant because it suggests that the warhead itself can act as a selectivity determinant. Consistent with this notion are the interisozyme selectivity data. For example, relative to Cl-amidine, the inclusion of the ortho carboxylate in o-Cl-amidine actually decreases the selectivity for PADs 2 and 3 by 23 to 3-fold. In contrast, with o-F-amidine the selectivity trends are the reverse, as the kinact/KI values are increased by 2- to 3-fold for PADs 2 and 3 (to 24- and 27-fold, respectively) and 6-fold for PAD4. Given these results, o-Cl-amidine is a PAD1 and 4 selective inhibitor, whereas o-F-amidine is primarily a PAD1 selective inhibitor. This preference for PAD1 suggests that o-F-amidine will serve as a versatile tool that can be used to assign physiological functions to PAD1. Based on the crystallographic data, as well as mutagenesis studies, the enhanced potency and selectivity is likely due to interactions between the ortho carboxylate and the indole nitrogen of W347 of PAD4. In cellulo studies indicate that these two compounds are better than F- and Cl-amidine in their ability to inhibit histone deimination, trigger HL-60 cell differentiation, and potentiate the cell killing effects of doxorubicin in HL-60 cells in a synergist and dose dependent fashion. Finally, o-F-amidine induces apoptosis of inflammatory cells to a greater extent than Cl-amidine. Given these findings, the incorporation of a carboxylate at this position will likely prove to be an important feature of third and fourth generation PAD inhibitors. Future studies will focus on evaluating the efficacy of o-F and o-Cl-amidine in mouse models of RA, colitis, and cancer.

Experimental

Chemicals

Cl-amidine (1), F-amidine (2), and 2-fluoroacetamidine (3) were synthesized as previously described.41, 42 PADs 1 to 4 were purified as previously described.4, 43 Cell culture media, RPMI 1640, and fetal bovine serum were obtained from ThermoScientific (Pittsburgh, PA). All compounds were purified to ≥95% purity. Purity was assessed by HPLC.

2-Fluoro-N-propylacetamidine●HCl (4)

To a solution of ethyl 2-fluorothioacetimidate (174 mg, 1.0 mmol) in dry ethanol (3 mL) at 0 °C was added propylamine (59 mg, 1.0 mmol). After 10 min, the reaction was allowed to warm to rt and stirring was continued for an additional 50 min. The mixture was partitioned between ether (3 mL) and H2O (5 mL); the aqueous layer was separated and lyophilized to yield the title compound as an ofF-white solid (134 mg, 87%). 1H NMR (400 MHz, D2O) δ (ppm): 5.07 (d, JHF = 45.2 Hz, 2H), 3.10 (t, J = 7.2 Hz, 2H), 1.46 (q, J = 7.2 Hz, 2H), 0.73 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, D2O) δ (ppm): 162.21 (d, JCF = 19.8 Hz), 77.53 (d, JCF = 177.4 Hz), 43.65, 20.15, 10.30. HRMS (ESI) m/z calculated for [C5H12FN2 +] 119.0984, observed 119.0993.

N-benzyl-2-fluoroacetimidamide●HCl (5)

To a solution of ethyl 2-fluorothioacetimidate (174 mg, 1.0 mmol) in dry ethanol (3 mL) at 0 °C was added benzylamine (107 mg, 1.0 mmol). After 10 min, the reaction was allowed to warm to rt and stirring was continued for an additional 50 min. The mixture was partitioned between ether (3 mL) and H2O (5 mL); the aqueous layer was separated and lyophilized to yield the title compound as a white powder (147 mg, 73%). 1H NMR (300 MHz, D2O) δ (ppm): 7.29 (m, 5H), 5.18 (d, JHF = 45 Hz, 2H), 4.44 (s, 2H). 13C NMR (75 MHz, D2O) δ (ppm): 162.82 (d, JCF = 19.8 Hz) 133.84, 129.27, 128.67, 127.84, 77.85 (d, JCF = 177.45 Hz), 45.74. HRMS (ESI) m/z calculated for [C9H12FN2+] 167.0984, observed 167.0979.

2-Fluoro-N-phenylacetimidamide●HCl (6)

To a solution of ethyl 2-fluorothioacetimidate (174 mg, 1.0 mmol) in dry ethanol (3 mL) at 0 °C was added aniline (93 mg, 1.0 mmol). After 10 min, the reaction was allowed to warm to rt and stirring was continued for an additional 50 min. The mixture was partitioned between ether (3 mL) and H2O (5 mL); the aqueous layer was separated and lyophilized to yield the title compound as an ofF-white solid (150 mg, 80%). 1H NMR (300 MHz, D2O) δ (ppm): 7.50-7.35 (m, 3H), 7.25 (m, 2H), 5.36 (d, JHF = 44.7 Hz, 2H) 13C NMR (75 MHz, D2O) δ (ppm): 163.52 (d, JCF = 19.8 Hz) 133.28, 130.94, 127.56, 125.31, 77.86 (d, JCF = 179.1 Hz). HRMS (ESI) m/z calculated for [C8H10FN2+] 153.0820, observed 153.0832.

N1-benzoyl-(3-(2-fluoroacetimidamido))-L-phenylalanine amide●TFA (7)

Rink AM Amide resin (300 mg, 0.2 mmol) was treated twice with 5 mL of 20% piperidine (in DMF) for 20 min and subsequently washed with dry DMF (3 × 5 mL). Fmoc-(3-nitro)Phe-OH (100 mg, 0.23 mmol), HOTT (S-(1-oxido-2-pyridyl)-thio-N,N,N’,N’-tetramethyluronium hexafluorophosphate; 303 mg, 0.35 mmol), and triethylamine (0.064 mL, 0.46 mmol) were dissolved in dry DMF (3 mL) and allowed to react for 10 min before being added to the resin. The reaction mixture was rocked at rt for 3 h before the resin was filtered and washed with DMF (3 × 5 mL). The resin was treated with acetic anhydride (0.19 mL, 2.0 mmol) and triethylamine (0.56 mL, 4.0 mmol) in DMF (5 mL) for 2 h to cap any remaining amino groups on the resin. The resin was collected by filtration, washed with DMF (2 × 5 mL), and treated twice for 20 min with 5 mL of 20% piperidine in DMF. The resin was then washed with DMF (3 × 5 mL), resuspended in DMF (5 mL), and treated with benzoyl chloride (0.093 mL, 0.8 mmol) and triethylamine (0.22 mL, 1.6 mmol) and rocked at rt overnight. The resin was washed with DMF (3 × 5 mL) then treated overnight at rt with 2M tin(II)chloride (6 mL), collected by filtration and washed with DMF (3 × 5 mL). The resin was then treated overnight with a solution of ethyl 2-fluorothioacetimidate (128 mg, 0.8 mmol) in DMF (5 mL), then collected by filtration, washed with DMF (3 × 5 mL), ethanol (3 × 5 mL), and DCM (3 × 5 mL), and dried for 1 h under reduced pressure. The dry resin was treated three times (1 h each) with 5 mL of TFA/TIS/H2O (95/2.5/2.5). The filtrates were collected, combined, and concentrated. Cold ether (25 mL) was added to the remaining residue, and the resulting precipitate was collected by centrifugation and purified by RP-HPLC to yield the title compound as an off-white solid (16.5 mg, 18%). 1H NMR (400 MHz, D2O) δ (ppm): 7.52 (m, 3H), 7.36 (m, 4H), 7.14 (m, 2H), 5.28 (d, JHF = 44.4 Hz), 4.72 (dd, J = 5.6, 9.6 Hz, 1H), 3.26 (dd, J = 5.6, 14.0 Hz, 1H), 2.97 (dd, J = 10.0, 14.0 Hz, 1H). 13C NMR (100 MHz, D2O) δ (ppm): 175.80, 170.63, 163.26 (d, JCF = 19.1 Hz), 139.42, 132.61, 132.45, 132.29, 130.50, 130.38, 128.65, 127.03, 126.04, 123.99, 77.60 (d, JCF = 178.8 Hz), 54.71, 36.65. HRMS (ESI) m/z calculated for [C18H20FN4O2+] 343.1570, observed 343.1565.

N1-benzoyl-(3-(2-fluoroacetimidamido))-L-phenylglycine amide●TFA (8)

Rink AM Amide resin (300 mg, 0.2 mmol) was treated twice with 5 mL of 20% piperidine (in DMF) for 20 min and subsequently washed with dry DMF (3 × 5 mL). Bz-(3-NO2)Phg-OH (180 mg, 0.6 mmol), HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronoium hexafluorphosphate; 228 mg, 0.6 mmol), HOBt (92 mg, 0.6 mmol), and triethylamine (0.17 mL, 1.2 mmol) were dissolved in dry DMF (5 mL) and allowed to react for 10 min before being added to the resin. The reaction mixture was rocked at rt for 3 h before the resin was collected by filtration and washed with DMF (3 × 5 mL). The resin was rocked overnight at rt in 2M tin(II)chloride (6 mL), collected by filtration and washed with DMF (3 × 5 mL). The resin was then treated overnight with a solution of ethyl 2-fluorothioacetimidate (128 mg, 0.8 mmol) in DMF (5 mL), then collected by filtration, washed with DMF (3 × 5 mL), ethanol (3 × 5 mL), and DCM (3 × 5 mL), and dried for 1 h under reduced pressure. The dry resin was treated three times (1 h each) with 5 mL of TFA/TIS/H2O (95/2.5/2.5). The filtrates were collected, combined, and concentrated. Cold ether (25 mL) was added to the remaining residue, and the resulting precipitate was collected by centrifugation and purified by RP-HPLC to yield the title compound as a white powder (19.5 mg, 22%). 1H NMR (400 MHz, D2O) δ (ppm): 7.64 (m, 2H), 7.48 (m, 3H), 7.36 (m, 3H), 7.28 (m, 1H), 5.54 (s, 1H), 5.34 (d, JHF = 44.8 Hz, 2H). 13C NMR (100 MHz, D2O) δ (ppm): 175.11, 173.95, 170.42, 163.54 (d, JCF = 19.8 Hz), 138.40, 132.98, 132.60, 132.55, 131.21, 129.70, 128.76, 127.36, 125.97, 124.87, 120.11, 77.69 (d, JCF = 178.1 Hz), 57.63. HRMS (ESI) m/z calculated for [C17H18FN4O2+] 329.1414, observed 329.1417.

N1-benzoyl-(4-(2-fluoroacetimidamido))-L-phenylalanine amide●TFA (9)

Rink AM Amide resin (300 mg, 0.2 mmol) was treated twice with 5 mL of 20% piperidine (in DMF) for 20 min and subsequently washed with dry DMF (3 × 5 mL). Fmoc-(4-nitro)Phe-OH (100 mg, 0.23 mmol), HOTT (303 mg, 0.35 mmol), and triethylamine (0.064 mL, 0.46 mmol) were dissolved in dry DMF (3 mL) and allowed to react for 10 min before being added to the resin. The reaction mixture was rocked at rt for 3 h before the resin was filtered and washed with DMF (3 × 5 mL). The resin was treated with acetic anhydride (0.19 mL, 2.0 mmol) and triethylamine (0.56 mL, 4.0 mmol) in DMF (5 mL) for 2 h to cap any remaining amino groups on the resin. The resin was collected by filtration, washed with DMF (2 × 5 mL), and treated twice for 20 min with 5 mL of 20% piperidine (in DMF). The resin was then washed with DMF (3 × 5 mL), resuspended in DMF (5 mL), and treated with benzoyl chloride (0.093 mL, 0.8 mmol) and triethylamine (0.22 mL, 1.6 mmol) and rocked at rt overnight. The resin was washed with DMF (3 × 5 mL) then treated overnight at rt with 2M tin(II)chloride (6 mL), collected by filtration and washed with DMF (3 × 5 mL). The resin was then treated overnight with a solution of ethyl 2-fluorothioacetimidate (128 mg, 0.8 mmol) in DMF (5 mL), then collected by filtration, washed with DMF (3 × 5 mL), ethanol (3 × 5 mL), and DCM (3 × 5 mL), and dried for 1 h under reduced pressure. The dry resin was treated three times (1 h each) with 5 mL of TFA/TIS/H2O (95/2.5/2.5). The filtrates were collected, combined, and concentrated. Cold ether (25 mL) was added to the remaining residue, and the resulting precipitate was collected by centrifugation and purified by RP-HPLC to yield the title compound as an ofF-white solid. 1H NMR (400 MHz, CD3OD) δ (ppm): 7.76 (d, J = 8.3 Hz, 2H), 7.55-7.49 (m, 3H), 7.44 (m, 2H), 7.29 (d, J = 8.3 Hz, 2H), 5.43 (d, JHF = 45.2 Hz), 3.66-3.48 (m, 1H), 3.36 (dd, J = 4.8, 13.8 Hz, 1H), 3.09 (dd, J = 9.4, 13.5, Hz, 1H). 13C NMR (100 MHz, D2O) δ (ppm): 175.99, 170.18, 164.99 (d, JCF = 19.4 Hz), 140.78, 135.25, 133.15, 132.56, 129.74, 128.55, 126.66, 80.20, 78.42 (d, JCF = 179.9 Hz), 56.16, 38.93. HRMS (ESI) m/z calculated for [C18H20FN4O2+] 343.1570, observed 343.1570.

N1-benzoyl-4-((2-fluoro-1-iminoethyl)aminomethyl)-L-phenylalanine amide●TFA (10)

Rink AM Amide resin (300 mg, 0.186 mmol) was pre-swelled in DMF (5 mL) for 1h, filtered, washed with DMF (2 × 5 mL), treated with 20% piperidine (in DMF) (2 × 5 mL) for 20 min, and subsequently washed with dry DMF (3 × 5 mL). Fmoc-L-(4-amino(Boc)methyl)Phe-OH (384 mg, 0.744 mmol), HBTU (282 mg, 0.744 mmol), and HOBt (114 mg, 0.744 mmol) were dissolved in DMF (3 mL) and allowed to stand at rt for 10 min before N-methylmorpholine (0.4 M in DMF) (3.7 mL, 1.49 mmol) was added. The reaction mixture was rocked at rt for 3 h before the resin was filtered, washed with DMF (3 × 5 mL), and treated with 20% piperidine (in DMF) (2 × 5 mL) for 1 h and subsequently filtered and washed with DMF (3 × 5 mL). A mixture of benzoyl chloride (105 mg, 0.744 mmol) and N-methylmorpholine (0.4 M in DMF) (3.7 mL, 1.49 mmol) was added to the resin, and this suspension was rocked at rt. After 16 h, the resin was collected by filtration and washed with DMF (3 × 5 mL), ethanol (2 × 5 mL), and DCM (2 × 5 mL), and dried overnight under reduced pressure. The dry resin was treated twice with a mixture of TFA/TIS/H2O (95/2.5/2.5) at rt for 3 h. The resulting filtrates were collected, combined, and concentrated under a stream of nitrogen. Cold ether (20 mL) was added to the remaining residue, and the resulting precipitate was collected by centrifugation, and purified by RP-HPLC to afford N-α-benzoyl-L-(4-aminomethyl)phenylalanine amide. Ethyl fluoroacetimidate hydrochloride (10 mg, 0.073 mmol), dry triethylamine (0.004 mL, 0.029 mmol), and N-α-benzoyl-L-(4-aminomethyl)phenylalanine amide (6 mg, 0.015 mmol) were dissolved in dry DMF (1 mL), and the mixture was stirred under nitrogen for 16 h. Purification by RP-HPLC afforded the title compound as a hygroscopic white powder upon lyophilization. 1H NMR (400 MHz, CD3OD) δ (ppm): 7.63 (d, J = 7.7 Hz, 2H), 7.42 (t, J = 7.4 Hz, 1H), 7.33 (t, J = 7.6 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 5.13 (d, J = 45.4 Hz, 2H), 4.73 (dd, J = 5.5, 10.0 Hz, 1H, obtained in D2O, 500 Mhz), 4.40 s, 2H), 3.27 (dd, J = 5.4, 13.9 Hz, 1H, obtained in D2O, 400 Mhz), 2.94 (dd, J = 9.9, 13.9 Hz, 1H). 13C NMR (100 MHz, CD3OD) δ (ppm): 175.87, 170.78, 162.63 (d, JCF = 18.9 Hz), 137.13, 132.85, 132.42, 132.38, 129.78, 128.68, 127.80, 127.11, 77.62 (d, JCF = 177.3 Hz), 55.03, 45.16, 36.79. HRMS (ESI) m/z calculated for [C19H22FN4O2+] 357.1227, observed 357.1732.

N1-benzoyl-N4-(2-fluoro-1-iminoethyl)-1,4-diaminobutane●TFA (11)

To a solution of N1-benzoyl-1,4-diaminobutane●TFA (39 mg, 0.13 mmol) in dry methanol (3 mL) was added ethyl 2-fluorothioacetimidate (36 mg, 0.25 mmol) and Cs2CO3 (62 mg, 0.19 mmol). The reaction was allowed to stir at rt overnight before being quenched with TFA. The mixture was subsequently diluted with H2O and purified by RP-HPLC to yield the title compound as a clear colorless residue (46.5 mg, 100%). 1H NMR (300 MHz, D2O) δ (ppm): 7.60-7.30 (m, 5H), 5.07 (d, JHF = 44.7 Hz, 2H), 3.26 (m, 4H), 1.47 (m, 4H). 13C NMR 100 MHz, D2O) δ (ppm): 170.83, 162.34 (JCF = 19.8 Hz), 133.51, 131.97, 128.65, 126.82, 77.49 (JCF = 177.4 Hz), 41.61, 39.06, 25.67, 23.95. HRMS (ESI) m/z calculated for [C13H19FN3+] 252.1512, observed 252.1517.

N1-benzenesulfonyl-N4-(2-fluoro-1-iminoethyl)-1,4-diaminobutane●TFA (12)

To a solution of N1-benzenesulfonyl-1,4-diaminobutane●TFA (43 mg, 0.13 mmol) in dry methanol (3 mL) was added ethyl 2-fluorothioacetimidate (36 mg, 0.25 mmol) and Cs2CO3 (62 mg, 0.19 mmol). The reaction was allowed to stir at rt overnight before being quenched with TFA. The mixture was subsequently diluted with H2O and purified by RP-HPLC to yield the title compound as a clear colorless residue (41.5 mg, 80%). 1H NMR (300 MHz, D2O) δ (ppm): 7.72 (m, 2H) 7.51 (m, 3H), 5.07 (d, JHF = 44.7 Hz, 2H), 3.08 (t, J = 6.9 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H) 1.45 (m, 4H). 13C NMR (75 MHz, D2O) δ (ppm): 162.50 (d, JCF = 19.3 Hz), 138.36, 133.65, 129.68, 126.76, 77.69 (d, JCF = 177.45 Hz), 42.23, 41.66, 25.99, 23.94. HRMS (ESI) m/z calculated for [C12H19FN3O2S+] 288.1182, observed 288.1183.

N1-benzenesulfonyl-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide●TFA (13)

Rink AM Amide resin (500 mg, 0.36 mmol) was pre-swelled in DMF (15 mL) for 1h, filtered, washed with DMF (2 × 15 mL), treated with 20% piperidine (in DMF) (2 × 15 mL) for 20 min, and subsequently washed with dry DMF (3 × 15 mL). Fmoc-Orn(Dde)-OH (552 mg, 1.07 mmol), HBTU (404 mg, 1.07 mmol), and HOBt (163 mg, 1.07 mmol) were dissolved in DMF (5 mL) and allowed to stand at rt for 10 min before N-methylmorpholine (0.4 M in DMF) (5.3 mL, 2.13 mmol) was added. The reaction mixture was rocked at rt for 16 h before the resin was filtered, washed with DMF (3 × 5 mL), and treated with 20% piperidine (in DMF) (2 × 15 mL) for 1 h and subsequently filtered and washed with DMF (3 × 15 mL). A mixture of benzenesulfonyl chloride (215 mg, 1.42 mmol) and N-methylmorpholine (0.4 M in DMF) (7 mL, 2.84 mmol) was added to the resin and this suspension was rocked at rt. After 16 h, the resin was collected by filtration and washed with DMF (3 × 15 mL). The resin was treated with 2% hydrazine (in DMF) (15 mL) for 2 h, washed with DMF (3 × 15 mL), ethanol (2 × 15 mL), and DCM (2 × 15 mL), collected by filtration, and dried overnight under reduced pressure. Ethyl fluoroacetimidate hydrochloride (390 mg, 2.77 mmol) and dry triethylamine (0.38 mL, 2.77 mmol) were added to the resin suspended in DMF (15 mL) and the mixture was stirred under nitrogen for 16 h. The resin was collected by filtration and washed sequentially with DMF (3 × 15 mL), ethanol (2 × 15 mL), and DCM (2 × 15 mL). The resin was incubated three times with a mixture of TFA (10%) and TIS (1%) in DCM for 1 h each. The filtrates were collected, combined, and concentrated under a stream of nitrogen. Cold ether (20 mL) was added to the remaining residue and the resulting precipitate was collected by centrifugation. This precipitate was washed with cold ether (2 × 15 mL) and lyophilized. Purification by RP-HPLC afforded the title compound as a hygroscopic white powder upon lyophilization. 1H NMR (400 MHz, CD3OD) δ (ppm): 7.89 (d, J = 8.4 Hz, 2H), 7.65-7.55 (m, 3H), 5.27 (d, J = 45.4 Hz, 2H), 3.82 (m, 1H), 3.10 (m, 2H, obtained in D2O, 400 MHz), 1.81-1.59 (m, 4H). 13C NMR (100 MHz, CD3OD) δ (ppm): 175.68, 164.28 (d, JCF = 19.5 Hz), 141.86, 133.93, 130.24, 128.17, 78.96 (d, JCF = 177.9 Hz), 57.13, 42.78, 31.56, 24.73. HRMS (ESI) m/z calculated for [C13H20FN4O3S+] 331.1240, observed 331.1237.

N1-(4-carbonyl)imidazole-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide●TFA (14)

Rink AM Amide resin (300 mg, 0.2 mmol) was treated twice with 5 mL of 20% piperidine (in DMF) for 20 min and subsequently washed with dry DMF (3 × 5 mL). Fmoc-Orn(Dde)-OH (414 mg, 0.8 mmol), HBTU (303 mg, 0.8 mmol), HOBt (122 mg, 0.8 mmol), and triethylamine (0.22 mL, 1.6 mmol) were dissolved in dry DMF (5 mL) and allowed to react for 10 min before being added to the resin. The reaction mixture was rocked at rt for 3 h before the resin was filtered, washed with DMF (3 × 5 mL), treated twice with 5 mL of 20% piperidine (in DMF) for 20 min, and washed with dry DMF (3 × 5 mL). 1H-imidazole-4-carbonyl chloride●HCl (134 mg, 0.8 mmol) and triethylamine (0.22 mL, 1.6 mmol) were dissolved in dry DMF (5 mL) and added to the resin. After rocking overnight at rt, the resin was collected by filtration, washed with DMF (3 × 5 mL), and treated twice with 2% hydrazine (in DMF) (5 mL) for 1 h to remove the Dde-protecting group. A solution of ethyl 2-fluoroacetimidate●HCl (113 mg, 0.8 mmol) and triethylamine (0.22 mL, 1.6 mmol) in DMF (5 mL) was added to the resin, and the mixture was rocked at rt. After 16 h, the resin was collected by filtration, washed with DMF (3 × 5 mL), ethanol (3 × 5 mL), and DCM (3 × 5 mL), then dried under reduced pressure for 1 h. The dry resin was treated three times (1 h each) with 5 mL of TFA/TIS/H2O (95/2.5/2.5). The filtrates were collected, combined, and concentrated. Cold ether (25 mL) was added to the remaining residue, and the resulting precipitate was collected by centrifugation and purified by RP-HPLC to yield the title compound as a white powder (49 mg, 48%). 1H NMR (400 MHz, D2O) δ (ppm): 8.65 (s, 1H), 7.92 (s, 1H), 5.10 (d, JHF = 44.8 Hz, 2H), 4.33 (dd, J = 5.6, 8.4 Hz, 1H), 3.24 (t, J = 6.8 Hz, 2H), 1.89-1.58 (m, 4H). 13C NMR (100 MHz, D2O) δ (ppm): 175.77, 162.54 (d, JCF = 19.1 Hz), 158.74, 135.42, 126.44, 123.44, 120.75, 117.64, 114.74, 77.51 (d, JCF = 177.4 Hz), 53.38, 41.32, 27.95, 23.17. HRMS (ESI) m/z calculated for [C11H18FN6O2+] 285.1475, observed 285.1468.

N1-(3-carbonyl)pyrrole-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide●TFA (15)

N1-(3-carbonyl)pyrrole-L-ornithine amide●TFA (18 mg, 0.053 mmol) and ethyl 2-fluoroacetimidate (15 mg, 0.11 mmol), and Cs2CO3 (26 mg, 0.080 mmol) were dissolved in dry methanol (1 mL). The reaction was stirred at rt for 16 h, quenched with TFA, diluted with H2O, and purified by RP-HPLC to yield the title compound as a white solid (11 mg, 54%). 1H NMR (400 MHz, D2O) δ (ppm): 10.78 (br, 1H), 6.95 (s, 1H), 6.78 (s, 1H), 6.19 (s, 1H), 5.10 (d, JHF = 44.8 Hz, 2H), 4.30 (dd, J = 4.8, 8.4 Hz, 1H), 3.25 (t, J = 6.8 Hz, 2H), 1.95-1.59 (m, 4H). 13C NMR (100 MHz, D2O) δ (ppm): 176.89, 175.71, 162.53 (d, JCF = 19.1 Hz), 123.97, 123.69, 112.13, 109.63, 77.52 (d, JCF = 178.1 Hz), 52.91, 41.32, 28.05, 23.26. HRMS (ESI) m/z calculated for [C12H19FN5O2+] 284.1523, observed 284.1526.

N1-(2-hydroxy-5-nitro)benzoyl-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide●TFA (16)

To a solution of N1-(2-hydroxy-5-nitro)benzoyl-L-ornithine●TFA (13.7 mg, 0.033 mmol) in dry methanol (1 mL) was added ethyl 2-fluoroacetimidate (9.4 mg, 0.067 mmol) and Cs2CO3 (16.3 mg, 0.049 mmol). The reaction was stirred overnight at rt, quenched with TFA, diluted with H2O, and purified by RP-HPLC to yield the title compound as a white powder (14.7mg, 95%). 1H NMR (400 MHz, D2O) δ (ppm): 8.45 (d, J = 3.2 Hz, 1H), 8.05 (dd, J = 2.8, 9.2 Hz, 1H), 6.88 (d, J = 9.2 Hz, 1H), 5.11 (d, JHF = 44.8 Hz, 2H), 4.39 (dd, J = 5.6, 8.4 Hz, 1H), 3.27 (t, J = 6.8 Hz, 2H), 1.95-1.61 (m, 4H). 13C NMR (100 MHz, D2O) δ (ppm): 175.95, 166.95, 162.55 (d, JCF = 20.5 Hz), 139.85, 128.96, 125.98, 117.69, 117.13, 77.53 (d, JCF = 177.4 Hz), 53.32, 41.32, 28.31, 23.05. HRMS (ESI) m/z calculated for [C14H18FN5O5+] 356.1370, observed 356.1359.

N-α-(2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-L-ornithine amide (o-F-amidine) (17)

Rink Amide AM resin (300 mg, 0.186 mmol) was suspended in 20% piperidine (in DMF) (5 mL) and gently rocked at rt for 20 min. The suspension was filtered and treated with 20% piperidine once more. After 20 min, the resin was filtered and washed with DMF (3 × 5 mL). Fmoc-Orn(Boc)-OH (338 mg, 0.74 mmol), HBTU (282 mg, 0.74 mmol) and HOBt (114 mg, 0.74 mmol) were dissolved in DMF (5 mL) and N-methylmorpholine (0.164 mL, 1.49 mmol) was added. After 10 min, this solution was added to the resin followed by gentle rocking. After 3 h, the resin was filtered, washed with DMF (3 × 5 mL) and treated with 20% piperidine (2 × 5 mL). The resin was then filtered, washed with DMF (3 × 5 mL) and resuspended in DMF (5 mL). After addition of phthalic anhydride (110 mg, 0.74 mmol) and triethylamine (0.207 mL, 1.49 mmol), the mixture was rocked at rt for 16 h. The resin collected by filtration, washed with DMF (3 × 5 mL), ethanol (2 × 5 mL) and dichloromethane (2 × 5 mL), dried under reduced pressure, and treated twice with a mixture of TFA/TIS/H2O (95/2.5/2.5) for 1 h. The filtrates were collected, combined, and concentrated under a stream of nitrogen. Cold ether (20 mL) was added to the remaining residue, and the resulting precipitate was collected by centrifugation, washed twice with ether (10 mL), and lyophilized. This precipitate (15 mg, 0.038 mmol), ethyl chloroacetimidate hydrochloride (10.7 mg, 0.076 mmol), and cesium carbonate (18.5 mg, 0.057 mmol) were dissolved in dry methanol (1 mL) and stirred at rt for 16 h. The reaction was quenched with TFA, diluted with H2O, and purified by RP-HPLC to afford the title compound as a hygroscopic white powder (15 mg, 23%). 1H NMR (400 MHz, D2O) δ (ppm): 7.83 (m, 1H), 7.56-7.45 (m, 2H), 7.35 (m, 1H), 4.34 (m, 1H), 3.25 (m, 2H), 1.92-1.55 (m, 4H). 13C NMR (100 MHz, D2O) δ (ppm): 176.04, 173.00, 170.28, 162.79, 135.99, 135.10, 132.56, 131.51, 130.41, 128.62, 127.39, 53.32, 41.87, 39.05, 27.89, 23.04. HRMS (ESI) m/z calculated for [C15H20ClN4O4+] 355.1173, observed 355.1172.

N-α-(2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (o-Cl-amidine) (18)

Rink Amide AM resin (300 mg, 0.186 mmol) was suspended in 20% piperidine (in DMF) (5 mL) and gently rocked at rt for 20 min. The suspension was filtered and treated with 20% piperidine once more. After 20 min, the resin was filtered and washed with DMF (3 × 5 mL). Fmoc-Orn(Boc)-OH (338 mg, 0.74 mmol), HBTU (282 mg, 0.74 mmol) and HOBt (114 mg, 0.74 mmol) were dissolved in DMF (5 mL) and N-methylmorpholine (0.164 mL, 1.49 mmol) was added. After 10 min, this solution was added to the resin followed by gentle rocking. After 3 h, the resin was filtered, washed with DMF (3 × 5 mL) and treated with 20% piperidine (2 × 5 mL). The resin was then filtered, washed with DMF (3 × 5 mL) and resuspended in DMF (5 mL). After addition of phthalic anhydride (110 mg, 0.74 mmol) and triethylamine (0.207 mL, 1.49 mmol), the mixture was rocked at rt for 16 h. The resin collected by filtration, washed with DMF (3 × 5 mL), ethanol (2 × 5 mL) and dichloromethane (2 × 5 mL), dried under reduced pressure, and treated twice with a mixture of TFA/TIS/H2O (95/2.5/2.5) for 1 h. The filtrates were collected, combined, and concentrated under a stream of nitrogen. Cold ether (20 mL) was added to the remaining residue, and the resulting precipitate was collected by centrifugation, washed twice with ether (10 mL), and lyophilized. This precipitate (11.0 mg, 0.028 mmol), ethyl fluoroacetimidate hydrochloride (8.8 mg, 0.056 mmol), and cesium carbonate (13.7 mg, 0.042 mmol) were dissolved in dry methanol (1 mL) and stirred at rt for 16 h. The reaction was quenched with TFA, diluted 2-fold with H2O, and purified by RP-HPLC to afford the title compound as a hygroscopic white powder (18 mg, 29%) 1H NMR (400 MHz, D2O) δ (ppm): 7.84 (m, 1H), 7.56-7.44 (m, 2H), 7.39 (m, 1H), 5.12 (d, JHF = 44.8 Hz, 2H), 4.34 (m, 1H), 3.25 (m, 2H), 1.92-1.59 (m, 4H). 13C NMR (100 MHz, D2O) δ (ppm):176.01, 172.92, 169.70, 162.56 (d, JCF = 19.8 Hz), 136.19, 132.85, 131.72, 130.39, 128.65, 127.42, 77.53 (d, JCF = 177.4 Hz), 53.35, 41.32, 27.90, 23.18. HRMS (ESI) m/z calculated for [C15H20FN4O4+] 339.1468, observed 339.1468.

Ethyl 2-fluorothioacetimidate (19)

2-Fluorooacetonitrile (561 mg, 9.5 mmol) and ethanethiol (1180 mg, 19 mmol) were added to 15 mL of 1M HCl (in ether) at 0 °C. The reaction was stirred overnight at 4 °C. The product, which precipitated from the solution, was collected by centrifugation, washed with cold ether (3 × 10 mL), and dried under vacuum for 3 h to yield the title compound as a white crystalline compound (926 mg, 56%).

N1-benzoyl-3-(2-fluoroacetimidamido)-L-phenylalanine amide●TFA (20)

Rink AM Amide resin (300 mg, 0.213 mmol) was pre-swelled in DMF (5 mL) for 1h, filtered, washed with DMF (2 × 5 mL), treated with 20% piperidine (in DMF) (2 × 5 mL) for 20 min, and subsequently washed with dry DMF (3 × 5 mL). Fmoc-Phe(3-NO2)-OH (368 mg, 0.852 mmol), HBTU (323 mg, 0.852 mmol), and HOBt (130 mg, 0.852 mmol) were dissolved in DMF (2.3 mL) and allowed to stand at rt for 10 min before N-methylmorpholine (0.4 M in DMF) (4.2 mL, 1.70 mmol) was added. The reaction mixture was rocked at rt for 3 h before the resin was filtered, washed with DMF (3 × 5 mL), and treated with 20% piperidine (in DMF) (2 × 5 mL) for 1 h and subsequently filtered and washed with DMF (3 × 5 mL). A mixture of benzoyl chloride (0.1 mL, 0.852 mmol) and N-methylmorpholine (0.4 M in DMF) (4.3 mL, 1.70 mmol) was added to the resin, and this suspension was rocked at rt. After 16 h, the resin was collected by filtration and washed with DMF (3 × 5 mL), rocked in a solution of tin(II)chloride (6 mL, 2M in DMF) for 24 h, collected by fitration, washed with DMF (3 × 5 mL), ethanol (2 × 5 mL), and DCM (2 × 5 mL), and dried overnight under reduced pressure. The resin was resuspended in dry DMF (5 mL) and treated with ethyl fluoroacetimidate hydrochloride (282 mg, 2 mmol) and dry triethylamine (0.28 mL, 2 mmol) under nitrogen for 16 h and subsequently collected by filtration and washed with DMF (3 × 5 mL), ethanol (2 × 5 mL), and DCM (2 × 5 mL) and dried under reduced pressure. Cleavage from the resin was accomplished by treatment with a mixture of TFA (10%) and TIS (1%) in DCM (10 mL) for 10 min. This treatment was repeated, and both filtrates were collected, combined and concentrated under a stream of nitrogen. Cold ether (20 mL) was added to the remaining residue, and the resulting precipitate was collected by centrifugation, washed twice with ether (10 mL), lyophilized, and purified by RP-HPLC to afford the title compound as a hygroscopic white powder upon lyophilization. 1H NMR (400 MHz, CD3OD) δ (ppm): 7.76 (d, J = 8.3 Hz, 2H), 7.55-7.49 (m, 3H), 7.44 (m, 2H), 7.29 (d, J = 8.3 Hz, 2H), 5.43 (d, J = 45.2 Hz, 2H), 3.66-3.48 (m, 1H), 3.36 (dd, J = 4.8, 13.8 Hz, 1H), 3.09 (dd, J = 9.4, 13.5 Hz, 1H). 13C NMR (100 MHz, CD3OD) δ (ppm): 175.99, 170.18, 165.00 (d, JCF = 19.3 Hz), 140.78, 135.25, 133.15, 132.56, 129.74, 128.55, 126.66, 79.31 (d, JCF = 176.6 Hz), 56.16, 38.93. HRMS (ESI) m/z calculated for [C18H20FN4O2+] 343.1570, observed 343.1570.

N-benzoyl-(m-nitro)phenylglycine (21)

To a solution of m-nitrophenylglycine44 (500 mg, 2.55 mmol) and NaHCO3 (715 mg, 675 mmol) in H2O (13 mL) and 1,4-dioxane (2.8 mL) was added benzoyl chloride (358 mg, 2.55 mmol in 1,4-dioxane (2.8 mL)), and the reaction was stirred at rt. After 2.5 h, the reaction was diluted with H2O (80 mL), acidified with concentrated HCl (pH ~1.0), and extracted with ether (3 × 100 mL). The organics were combined, washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure. The remaining residue crystallized upon the addition of ether, and the product was collected by filtration as a white powder (575 mg, 75%). 1H NMR (300 MHz, CD3OD) δ (ppm): 8.42 (m, 1H), 8.27-8.18 (m, 1H), 7.95-7.85 (m, 3H), 7.67-7.41 (m, 4H), 5.87 (s, 1H). 13C NMR (75 MHz, CD3OD): δ (ppm): 171.14, 168.48, 139.38, 134.00, 133.46, 131.68, 129.52, 128.74, 128.20, 127.24, 123.29, 122.68, 122.39. HRMS (ESI) m/z calculated for [C15H13N2O5+] 301.0824, observed 301.0824.

N1-Boc-N4-benzoyl-1,4-diaminobutane (22)

To a solution of N1-Boc-1,4-diaminobutane●HCl (50 mg, 0.22 mmol) and Na2CO3 (70 mg, 0.66 mmol) in a mixture of H2O (1 mL) and 1,4-dioxane (0.25 mL) was added benzoyl chloride (0.027 mL, 0.23 mmol). After stirring overnight at rt, the reaction mixture was partitioned between ether (10 mL) and 1M HCl (5 mL). The organic layer was subsequently separated and washed with 5 mL portions of 1M HCl, H2O, and brine, dried over MgSO4, and concentrated under reduced pressure to yield the title compound as a white solid (60.8 mg, 95%). 1H NMR (400 MHz, CDCl3) δ (ppm): 7.73 (d, J = 7.2 Hz, 2H), 7.40 (m, 1H), 7.33 (m, 2H), 6.69 (br, 1H), 4.71 (br, 1H), 3.39 (q, J = 6.4 Hz, 2H), 3.07 (q, J = 6.4 Hz, 2H), 1.61-1.45 (m, 4H), 1.36 (s, 9H). 13C NMR (75 MHz, CDCl3): δ (ppm): 167.65, 156.24, 134.60, 131.32, 128.46, 126.97, 79.21, 39.62, 28.39, 27.67, 26.58. HRMS (ESI) m/z calculated for [C16H25N2O3+] 293.1865, observed 293.1860.

N1-benzoyl-1,4-diaminobutane●TFA (23)

Cold TFA (3 mL) was added to a reaction vial containing N1-Boc-N4-benzoyl-1,4-diaminobutane (46.4 mg, 0.16 mmol) and the mixture stirred at 0 °C. After 30 min, the reaction was allowed to warm to rt and was stirred for an additional 30 min before the excess TFA was removed. The resulting residue was dissolved in H2O (6 mL), washed with ether (3 mL) and lyophilized to yield the title compound as a clear colorless residue (47.9 mg, 98%). 1H NMR (400 MHz, D2O) δ (ppm): 7.58 (m, 2H), 7.42 (m, 1H), 7.35 (m, 2H), 3.21 (t, J = 6.4 Hz, 2H), 2.85 (t, J = 7.0 Hz, 2H), 1.58 (m, 4H). 13C NMR (75 MHz, D2O) δ (ppm): 170.82, 133.47, 131.96, 128.63, 126.83, 38.99, 25.43, 24.11. HRMS (ESI) m/z calculated for [C11H17N2O+] 193.1341, observed 193.1335.

N1-Boc-N4-benzenesulfonyl-1,4-diaminobutane (24)

To a solution of N10Boc-1,4-diaminobutane●HCl (50 mg, 0.22 mmol) and Na2CO3 (70 mg, 0.66 mmol) in a mixture of H2O (1 mL) and 1,4-dioxane (0.25 mL) was added benzenesulfonyl chloride (0.029 mL, 0.23 mmol) in 1,4-dioxane (0.25 mL). After stirring overnight at rt, the reaction mixture was partitioned between ether (10 mL) and 1M HCl (5 mL). The organic layer was subsequently separated and washed with 5 mL portions of 1M HCl, H2O, and brine, dried over MgSO4, and concentrated under reduced pressure to yield the title compound as a clear colorless residue (61.5 mg, 85%). 1H NMR (400 MHz, CDCl3) δ (ppm): 7.85 (m, 2H), 7.51 (m, 1H), 7.45 (m, 2H), 5.33 (br, 1H), 4.58 (br, 1H), 2.95 (m, 2H), 2.85 (q, J = 6.4 Hz, 2H), 1.41 (m, 4H), 1.38 (s, 9H). 13C NMR (75 MHz, CDCl3): δ (ppm): 156.10, 139.91, 132.56, 129.09, 127.20, 126.98, 79.20, 42.80, 39.86, 28.38, 27.10, 26.66. HRMS (ESI) m/z calculated for [C15H25N2O4S+] 329.1535, observed 329.1532.

N1-benzenesulfonyl-1,4-diaminobutane●TFA (25)

Cold TFA (3 mL) was added to a reaction vial containing N4-Boc-N1-sulfonyl-1,4-diaminobutane (54.5 mg, 0.17 mmol) and the mixture stirred at 0 °C. After 30 min, the reaction was allowed to warm to rt and was stirred for an additional 30 min before the excess TFA was removed. The resulting residue was dissolved in H2O (6 mL), washed with ether (3 mL) and lyophilized to yield the title compound as a clear colorless residue (50.9 mg, 87%). 1H NMR (400 MHz, D2O) δ (ppm): 7.69 (m, 2H), 7.54 (m, 1H), 7.43 (m, 2H), 2.79 (m, 4H), 1.49 (m, 2H), 1.35 (m, 2H). 13C NMR (75 MHz, D2O) δ (ppm): 137.88, 133.44, 129.44, 126.51, 41.87, 38.83, 25.56, 23.80. HRMS (ESI) m/z calculated for [C10H17N2O2S+] 229.1010, observed 229.1010.

N1-(2-hydroxy-5-nitro)benzoyl-L-ornithine●TFA (26)

Rink AM Amide resin (300 mg, 0.2 mmol) was treated twice with 5 mL of 20% piperidine (in DMF) for 20 min and subsequently washed with dry DMF (3 × 5 mL). Fmoc-Orn(Boc)-OH (364 mg, 0.8 mmol), HBTU (303 mg, 0.8 mmol), HOBt (122 mg, 0.8 mmol), and triethylamine (0.22 mL, 1.6 mmol) were dissolved in dry DMF (5 mL) and allowed to react for 10 min before being added to the resin. The reaction mixture was rocked at rt for 3 h before the resin was filtered, washed with DMF (3 × 5 mL), treated twice with 5 mL of 20% piperidine (in DMF), and washed with dry DMF (3 × 5 mL). 5-Nitrosalicylic acid (147 mg, 0.8 mmol), HBTU (303 mg, 0.8 mol), HOBt (122 mg, 0.8 mmol), and triethylamine (0.22 mL, 1.6 mmol) were dissolved in dry DMF (5 mL) and allowed to react for 10 min before being added to the resin. The reaction mixture was rocked at rt for 6 h. The resin was filtered and washed with DMF (3 × 5 mL), ethanol (3 × 5 mL), and DCM (5 mL). The resin was dried under reduced pressure for 1 h prior to being treated three times with 5 mL of TFA/TIS/H2O (95/2.5/2.5) for 1 h. The filtrates were collected, combined, and concentrated prior to the addition of ether (25 mL). The resulting precipitate was collected by centrifugation and washed with an additional portion of ether. The crude product was purified by RP-HPLC to yield the title compound as a yellow powder (15.1 mg, 18%). 1H NMR (400 MHz, CD3OD) δ (ppm): 8.89 (d, J = 2.8 Hz, 1H), 8.29 (dd, J = 2.4, 9.2 Hz, 1H), 7.09 (d, J = 9.2 Hz, 1H), 4.70 (dd, J = 5.2, 8.4 Hz, 1H), 2.98 (m, 2H), 2.09 (m, 1H), 1.91 (m, 1H), 1.78 (m, 2H). 13C NMR (100 MHz, CD3OD) δ (ppm): 174.37, 166.60, 163.98, 140.16, 128.39, 125.47, 117.64, 116.61, 52.45, 38.84, 28.97, 23.59. HRMS (ESI) m/z calculated for [C12H17N4O5+] 297.1199, observed 297.1207.

N1-(3-carbonyl)pyrrole-L-ornithine amide●TFA (27)

Rink AM Amide resin (300 mg, 0.2 mmol) was treated twice with 5 mL of 20% piperidine (in DMF) for 20 min and subsequently washed with dry DMF (3 × 5 mL). Fmoc-Orn(Boc)-OH (364 mg, 0.8 mmol), HBTU (303 mg, 0.8 mmol), HOBt (122 mg, 0.8 mmol), and triethylamine (0.22 mL, 1.6 mmol) were dissolved in dry DMF (5 mL) and allowed to react for 10 min before being added to the resin. The reaction mixture was rocked at rt for 3 h before the resin was filtered, washed with DMF (3 × 5 mL), treated twice with 5 mL of 20% piperidine (in DMF), and washed with dry DMF (3 × 5 mL). 1H-pyrrole-3-carboxylic acid (89 mg, 0.8 mmol), HBTU (303 mg, 0.8 mol), HOBt (122 mg, 0.8 mmol), and triethylamine (0.22 mL, 1.6 mmol) were dissolved in dry DMF (5 mL) and allowed to react for 10 min before being added to the resin. The reaction mixture was rocked at rt for 6 h. The resin was filtered and washed with DMF (3 × 5 mL), ethanol (3 × 5 mL), and DCM (5 mL). The resin was dried under reduced pressure for 1 h prior to being treated three times with 5 mL of TFA/TIS/H2O (95/2.5/2.5) for 1 h. The filtrates were collected, combined, and concentrated prior to the addition of ether (25 mL). The resulting precipitate was collected by centrifugation and washed with an additional portion of ether. The crude product was purified by RP-HPLC to yield the title compound as an ofF-white solid (19.5 mg, 29%). 1H NMR (400 MHz, D2O) δ (ppm): 10.7 (br, 1H), 6.91 (s, 1H), 6.75 (s, 1H), 6.11 (s, 1H), 4.27 (dd, J = 5.2, 9.2 Hz, 1H), 2.85 (t, J = 7.2 Hz, 2H), 1.91-1.51 (m, 4H). 13C NMR (100 MHz, D2O) δ (ppm): 176.70, 175.37, 123.94, 123.38, 112.04, 109.60, 52.81, 38.71, 27.80, 23.37. HRMS (ESI) m/z calculated for [C10H17N2O2+] 225.1351, observed 225.1346.

N1-(2-carboxy)benzoyl-N5-Boc-L-ornithine amide●TFA (28)

To a solution of H-Orn(Boc)-OH (232 mg, 1.0 mmol) and triethylamine (0.14 mL, 1.0 mmol) in H2O (1 mL) was added phthalic anhydride (148 mg, 1.0 mmol, dissolved in 3 mL of THF). A second portion of triethylamine (0.14 mL, 1.0 mmol) was added and the reaction was stirred at rt for 2 h. The THF was removed under reduced pressure and the remaining solution was acidified with concentrated HCl to pH ~ 1. (Note: Acidification results in the precipitation of a white solid). The acidified mixture was extracted with DCM (3 × 3 mL). The organics were combined, washed with H2O (2 × 2 mL) and brine (2 mL), dried over MgSO4, and concentrated to afford the title compound as a white powder (300 mg, 79%). 1H NMR (300 MHz, CD3OD) δ (ppm): 7.95 (m, 1H), 7.65-7.45 (m, 3H), 4.59 (m, 1H), 3.12 (m, 2H), 2.05-1.55 (m, 4H), 1.4 (s, 9H). 13C NMR (75 MHz, CD3OD) δ (ppm): 173.96, 171.29, 167.97, 157.20, 131.64, 130.66, 129.84, 129.60, 129.24, 127.60, 78.58, 52.51, 39.48, 28.43, 27.39, 25.88. HRMS (ESI) m/z calculated for [C18H25N2O7+] 381.1662, observed 381.1668.

IC50 Values. IC50 values were measured as previously described.21, 45, 46 Briefly, PAD4 (0.2 μM) was pre-incubated with varying concentrations of inhibitor in Reaction Buffer Buffer (100 mM Tris-HCl pH 7.6, 10 mM CaCl2, 2 mM DTT, and 50 mM NaCl) for 15 min at 37 °C. BAEE (10 mM) was added and the reaction was allowed to proceed for 15 min at 37 °C. The initial rate data were fit to eq 1,

equation M1
(1),

using Grafit 5.0.1.1.47

Reversibility of Inhibition

To demonstrate that o-F- and o-Cl-amidine irreversibly inactivate all four PAD isozymes, PAD1 (2 μM), PAD2 (5 μM), PAD3 (5 μM), and PAD4 (2 μM) were treated with excess inhibitor (1 mM final) for 1 h. The reactions were then dialyzed for 20 h against buffer (2 L) containing 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 mM DTT, 500 mM NaCl, and 10% glycerol. Residual activity was then measured using our standard citrulline production assay.2, 48

Substrate Protection

To demonstrate that substrate protects against the inactivation by o-F- and o-Cl-amidine, reaction mixtures containing 100 mM Tris-HCl pH 7.6, 2 mM DTT, 50 mM NaCl, and 10 mM CaCl2, and 10 mM or 2 mM BAEE were pre-incubated with and without inactivator for 10 min. Enzyme (PAD1 (0.2 μM), PAD2 (0.5 μM), PAD3 (0.5 μM), or PAD4 (0.2 μM)) was added and aliquots were removed at different time points (0-15 min for PAD1, PAD2, and PAD4 and 0-30 min for PAD3). The amount of product formed was measured as previously described 2, 48 and the data were fit to eq 2,

equation M2
(2),

using GraFit (version 5.0.11),47 where vi is the initial velocity, kobs·app is the apparent pseudo first order rate constant for inactivation, t is time, and P is amount of product formed during the reaction.

Inactivation kinetics

The inactivation kinetic parameters were determined by incubating PAD1, 2, 3, or 4 in an inactivation mixture containing 10 mM CaCl2, 2 mM DTT, and 100 mM Tris-HCl pH 7.6 and aliquots were taken out at different time points from 0-30 min. These aliquots were added to a reaction mixture containing 100 mM Tris-HCl pH 7.6, 10 mM CaCl2, 2 mM DTT, 50 mM NaCl, and 10 mM BAEE. The reactions were quenched in liquid nitrogen after a 15 min incubation. The residual activity was then measured at each of the different concentrations of inactivator as a function of time, and the data were fit to eq 3,

equation M3
(3),

using GraFit version 5.0.11,47 where v is the velocity, vo is the initial velocity, k is the pseudo-first order rate constant of inactivation (i.e., kobs), and t is time. The kobs values were then plotted versus inactivator concentration, and, for the case where saturation was achieved, the data were fit to eq 4,

equation M4
(4),

using GraFit version 5.0.11.47 kinact corresponds to the maximal rate of inactivation and KI is the concentration of I that yields halF-maximal inactivation. If saturation was not seen, and the plot of kobs versus [I] was linear, then the slope of the line corresponded to kinact/KI.

Cell culture and cytotoxicity assay

HL60 human promyelocytic leukemia cells were cultured in RPMI 1640 media, supplemented with 10% FBS and 1% Pencillin-Streptomycin. Cells were grown in a 5% CO2 incubator at 37 °C. HL-60 cells were grown to a confluence of 1 × 106 cells/mL in the appropriate media. 90 QL of the cells were added to each well of a 96 well plate. o-F-amidine, o-Cl-amidine or doxorubicin, (10 QL, 100 nM to 100 μM final) were added to the plates and allowed to incubate with the cells for 24 h. Cell viability was determined using the CellTiter 96 Non-radioactive Cell Proliferation Assay (Promega). Cell viability was quantified as the percentage of control absorbance. Each condition was performed in triplicate. The use of 1% Triton X-100 served as a 100% killing control. When possible, EC50 values were determined by fitting the dose response data to equation 1 using GraFit (version 5.0.11).47 Here, [I] is the concentration of inhibitor (e.g., doxorubicin) and EC50 is the concentration of inhibitor that yields halF-maximal cell survival.

Histone deimination

HL60 cells (1×106 ml/cell) were treated with ATRA (1 μM final concentration) for 48 h at 37 °C, 5% CO2. Cells were split into 12 well plates and treated with 2 mM CaCl2 and either o-F-amidine, o-Cl-amidine, or Cl-amidine (1 μM to100 μM). After 15 min at 37 °C in an atmosphere containing 5% CO2, the calcium ionophore A23187 (4 μM) was added. Cells were harvested after 15 min treatment with the stimuli, rinsed with cold PBS and lysed with SDS lysis buffer (2% SDS, 62.5 mM Tris, pH 6.8, 10% glycerol). Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot analysis. Membranes were blocked with 5% nonfat dried milk in TBST for 1 h at room temperature, and subsequently probed with polyclonal anti-citrulline H3 antibody (Abcam, ab5103) or polyclonal anti-actin (Abcam, ab1801).

Cell Culture and Treatment

TK6 lymphoblastoid cells were a kind gift from Curtis C. Harris (National Cancer Institute, Bethesda, MD), originally derived from Dr. William Thilly’s and Howard Liber’s labs. TK6 cells are a lymphoblastoid cell line derived from the spleen more than 30 y ago. TK6 cells were maintained in exponentially growing suspension culture at 37°C in a humidified 5% CO2 atmosphere in RPMI 1640 supplemented with 10% heat-inactivated calf serum, 100 units/mL penicillin, 100 Wg/mL streptomycin, and 2 mmol/L l-glutamine. 12 hours before treatment, media was changed to that above, except supplemented with 1% heat-inactivated calf serum. Treatment was with 50 μg/ml of Cl-amidine or o-F-amidine for 0 – 24 hours as indicated.

Apoptosis

Apoptosis was measured by Annexin V as previously described.27 PARP cleavage was measured by standard western blot procedures as described previously.49 The antibodies were anti-PARP (Cell Signaling Technology, Cat#9542) and GAPDH (Abcam, ab9484).

Crystallization of PAD4 inhibitor complexes and structure determination

Crystals of o-F-amidine or o-Cl-amidine in complex with wild-type PAD4 were prepared by soaking the compound into crystals of wild-type PAD4. Crystals of the wild-type enzymes were prepared according to previously established methods.50, 51 Subsequently, these crystals were transferred to fresh crystallization buffer containing 5mM CaCl2 and 5mM o-F-amidine or o-Cl-amidine for 6h. Diffraction data were then collected on a beamline BL-17A at Photon Factory (Tsukuba, Japan), and then scaled using the program HKL2000.52 The initial structure of the PAD4·o-F-amidineecalcium complex or PAD4·o-Cl-amidine·calcium complex was derived from the atomic coordinates of the PAD4(C645A)·BAA·calcium complex (Protein Data Bank entry: 1WDA). The structures were manually improved using the program COOT.53 At this stage, the inhibitor moiety in the complex was identified on the |Fo| - |Fc| electron density maps. The structures were refined by the programs CNS54 and REFMAC.55 Crystallographic data were given in Table 1. Final coordinates and structure factors have been deposited to the Protein Data Bank Japan (PDBj). The PDB identification numbers are 1B1U and 1B1T for the PAD4•o-F-amidine and PAD4•o-Cl-amidine complexes, respectively.

Table 1
Crystallographic data and refinement statistics

Supplementary Material

supplement

Abbreviations

PAD
protein arginine deiminase
Cit
citrulline
RA
rheumatoid arthritis
BAEE
benzoyl-L-arginine ethyl ester
BAEE
benzoyl-L-arginine ethyl ester
BAA
benzoyl-L-arginine amide
BAME
benzoyl-L-arginine methyl ester
DTT
dithiothreitol
TCEP
tris-2-carboxyethyl phosphine
EDTA
ethylenediaminetetraacetic acid
PTM
post-translational modification
NET
neutrophil extracellular trap
DSS
dextran sodium sulfate
HOTT
S-(1-oxido-2-pyridyl)-thio-N,N,N’,N’-tetramethyluronium hexafluorophosphate
HBTU
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronoium hexafluorphosphate
TIS
triisopropyl silane

Footnotes

This work was supported in part by funds from the University Of South Carolina (P.R.T), The Scripps Research Institute, and by National Institutes of Health grants GM079357 (P.R.T.) and CA151304 (P.R.T. and L.J.H).

The coordinates for the PAD4•o-F-amidine and PAD4•o-Cl-amidine complexes have been deposited in the PDBJ. The PDB identification numbers are 1B1U and 1B1T, respectively.

Supporting Information Available

Supplementary Figures S1-S2. This material is available free of charge via the Internet at http://pubs.acs.org.

References

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