Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Biarylsulfonamide CCR9 inhibitors for inflammatory bowel disease Jing Zhang ⇑, Jan Romero, Audrey Chan, Jennifer Goss, Sabrina Stucka, Jason Cross, Brian Chamberlain, Mustafa Varoglu, Haoqun Chandonnet, Dominic Ryan, Blaise Lippa Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, United States
a r t i c l e
i n f o
Article history: Received 14 May 2015 Revised 10 June 2015 Accepted 12 June 2015 Available online xxxx
a b s t r a c t Inflammatory bowel disease, including Crohn’s disease and ulcerative colitis, affects millions of people worldwide. CCR9 has been shown to be a key chemokine receptor mediating the local inflammatory responses in the GI tract. The CCR9 inhibitor Vercirnon advanced to phase 3 clinical trials, but carries several liabilities which we sought to improve. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: IBD Crohn’s disease Ulcerative colitis CCR9 Sulfonamide
Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic inflammation in the gastrointestinal tract. It affects millions of people worldwide with >1 million new cases each year in the US alone. Despite extensive research, current therapeutic options for IBD are still very limited. Steroids are highly effective in achieving remission but long term use leads to significant side effects and drug tolerance. Immunosuppressants are often applied to maintain remission, but are sub-optimal in terms of efficacy and safety. In the last 10 years, the standard of care in the US has become antiTNF monoclonal antibodies (e.g. infliximab) in combination with the immunosuppressant azathioprine. About two thirds of the patients initially respond to this therapy, but only 50% remain in remission after a year of therapy. Thus safer and more effective therapeutic strategies for IBD are needed. Although the etiology of IBD has not been fully elucidated, genetic, immune and environmental risk factors have been implicated. Biopsy samples from patients with active disease show large numbers of leukocyte, lymphocyte and monocyte infiltrations in the intestinal/colonic mucosa. Data from a variety of immuneengineered IBD mouse models also suggest that chronic gastrointestinal tract inflammation results from a dysregulated immune response to otherwise harmless commensal flora.1–4 The adaptive immune system, particularly T cells, is believed to play a pivotal role in the induction and perpetuation of IBD pathogenesis.5,6
⇑ Corresponding author. Tel.: +1 781 640 2333. E-mail address: [email protected] (J. Zhang).
Chemokine receptors and their ligands regulate the migration and maturation of inflammatory and immune cells. CCR9 is a C– C chemokine receptor, expressed mainly in circulating lymphocytes. CCL25, the only identified ligand for CCR9, is primarily expressed in thymus and intestine which in turn results in enriched CCR9-positive cells in the intestine. Accumulating evidence indicates CCR9 expressing pro-inflammatory cells are involved in the IBD disease initiation and progression.7–10 Due to the high unmet medical need for IBD, and the strong biological validation of the CCR9/CCL25 pathway to mediate lymphocyte homing to the intestine, CCR9 has attracted a great deal of attention.11 Most notably, GSK/Chemocentryx was able to advance its CCR9 antagonist Vercirnon into a critical phase III clinical trial. Unfortunately, the clinical program was terminated in late 2013 due to insufficient efficacy in IBD. It is believed that the lack of clinical efficacy could have been due to Vercirnon’s poor physicochemical properties and inability to sufficiently inhibit CCR9 continuously. Most notably, the extremely poor solubility of Vercirnon has resulted in variable PO and SC PK in our laboratories. Structurally, it carries a biaryl ketone, which is a potential structural alert due to its reactivity. Herein we wish to report our efforts toward an improved CCR9 inhibitor based on the biarylsulfonamide scaffold (Fig. 1). Conformational analysis of Vercirnon shows that its ketone may form an intramolecular hydrogen bond with the sulfonamide NH, restricting the overall conformation of the biaryl ketone. The corresponding N–Me analog of Vercirnon abrogates the CCR9 binding affinity completely, supporting this conformational hypothesis, not but excluding other possibilities. Our strategy was to replace
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Please cite this article in press as: Zhang, J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.046
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J. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx O O S
Intramolecular HB O
NH
O
O S
N+
O-
NH
N
R
Cl
Vercirnon IC50 = 10 nM Chemotaxis (serum) = 539 nM logD = 3.7 LipE = 4.4 Solubility pH7.4/9.0 (ug/mL): 1.2/51
Cl
Figure 1. Vercirnon structure and design strategy.
O O S Cl
O O S NH
NH 2 Br +
a
Br
b
Cl O O S NH
B
O B
O
A
O O S NH
c
Cl
Ar
Cl
Cl
C
Scheme 1. General synthetic route. Reagents and conditions: (a) pyridine, 80 °C, (b) bis(pinacolato)diboron, Pd(dppf)2Cl2, KOAc, 1,4-dioxane, 100 °C, 50%; (c) ArBr, Pd(dppf)2Cl2, aq Na2CO3, 1,4-dioxane, 100 °C.
Table 2 SAR around the hetero-aryls
Table 1 Pyridine SAR
O O
O O S NH
1
S
6 5
N
NH
N
R
2
4 3
Cl
Cl
ID
R
CCR9 Ca2+ IC50 (nM)12
LipE
Chemotaxis (Buffer) IC50 (nM)
1 2 3 4 5 6 7 8 9
H 4-OMe 3-OMe 3-OH 3-NH2 3-CH2OH 3-CH2NH2 3-CH2OCH3 3-OH, Pyridine Noxide 3-OH, 4-CONH2 3-OH, 4-CH2OH
>10,000 2500 958 29 95 89 3163 3477 491
10000
2.9
—
29
2.9
1310
1000
0.3
—
55
3.2
—
29
3.2
23
26
3.8
23
N
1 N
4 OH N
12
N N
N
13 NH 2 N
the potentially problematic arylketone moiety with functionalized heterocycles, while retaining a nitrogen atom in the 20 -position to maintain the critical intramolecular hydrogen bonding motif. It was envisaged that suitably positioned solubilizing groups extending from the heterocycle would improve the overall physiochemical properties. The analog synthesis was straightforward as depicted in Scheme 1. We first explored 2-pyridyl as the biarylketone replacement (Table 1). Although the parent 2-pyridyl analog 1 was inactive toward CCR9, preliminary substituted pyridine SAR studies identified 3-methoxy-2-pyridyl analog 3 with 1 lM activity.
N
14
NH 2
N
N
15 NH 2
Interestingly, simply removing the methyl off the methoxy gave compound 4 with 29 nM CCR9 inhibition. Further analoging on the C3 position revealed that only small functional groups were
Please cite this article in press as: Zhang, J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.046
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J. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx Table 3 Pyrimidines SAR
Table 4 Pyrazines SAR O O S
NH
3 N 4
O O
2
N 5
R
S
1 6
2
CCR9 Ca2+ IC50 (nM)
R
N
Ca2+ LipE
Chemotaxis (Buffer) IC50 (nM)
ID
3.2
—
14
N
N
4.5
750
Chemotaxis (Buffer) IC50 (nM)
29
3.2
23
N
25
3.3
235
N
421
1.6
—
34
3.9
34
614
2.8
—
3604
2.5
—
812
5.9
—
268
2.7
—
1005
2.0
—
715
1.7
—
N
25
OH
HN
NH 2
N
N
4
5.0
30
26 HN
OH
N
NHMe
18
Ca2+ LipE
N
11
N
4
NH 2
16
17
3
CCR9 Ca2+ IC50 (nM)
R
NH 2
N
5
N
N
55
13 N
6R
Cl
Cl
ID
NH 1 N
N
27
N
27
4.0
HN
—
OH
N
OH
OH
HN
19
N
N
HN
214
3.9
14
NH 2
4.6
HN
N
N
30 HN
N
21
9
N
3.7
COOH
N
105
OH
N
31 HN
N
22
26
N NH2
3.9
N
32 11
5.6
HN
325
OH
Cl
OH N
N
6
24
H N
N
O OH
OH
190
N
23 N
N
N
340
OH
N
N
29
N
20
N
OH
N
—
OH N
N
28
3.7
60
OH
tolerated, and a hydrogen bond donor was critical for enzymatic activity. While 3-amino and 3-hydroxymethyl substituents were well tolerated (potency for 5 and 6 is within 3 of 3-hydroxyl analog 4), moderately basic functional groups such as 3-aminomethyl analog 7 was not tolerated. For initial SAR studies around the pyridine ring, we chose the 3-hydroxy analog as our next benchmark. Small substituents on pyridine positions 5 and 6 were tolerated, while position 4 could accommodate a variety of functional groups. We sought to improve the physicochemical properties via C4 substitutions. The 4-carboxylamide and 4-hydroxymethyl analogs 10 and 11 stood out as two of the best. Analog 11 had an enzymatic IC50 of 8.7 nM and a functional IC50 of 330 nM in the buffer migration assay. To further improve physiochemical properties and CCR9 potency, we briefly surveyed some other nitrogen-containing heterocycles (Table 2). Interestingly, 2-pyrimidine analog 12 improved
33
N HN
OH
the activity over the parent 2-pyridine by >10. The aminopyrimidine, aminopyrazine and aminopyridazine analogs 13–15 all displayed similar enzymatic potencies to the lead 3hydroxylpyridine analog 4. However, the aminopyrazine and aminopyridazine analogs 14 and 15 showed 50 improvement in CCR9 functional activity over 4. Due to synthetic feasibility and metabolic stability considerations, we decided to focus on the pyrimidine and pyrazine scaffolds for further SAR studies. Representative pyrimidine SAR is detailed in Table 3. As in the pyridine series, o-hydroxyl 16 gave better CCR9 inhibition than o-amino substituent. Small, polar substituents at the C2 position gave the best activity (17, 4 nM). As the size increases, the activity gradually deteriorates (18, 19). The C6 position tolerates a variety of functional groups in terms of size and polarity (21–24). Among them, 6-dimethylamino and 6-ethyl analogs 21 and 24
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J. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 5 In vitro/in vivo (mice) profiles of 27 versus Vercirnon Compd
Ca2+ IC50 (nM)
LipE
Chemotaxis (Buffer) IC50 (nM)
Chemotaxis (Serum) IC50 (nM)
Sol. pH 7.4/ 9 (lg/mL)
hPPB (% Bound)
HLM/MLM CL (ml/min/kg)
DN_AUC IV (lg hr/mL)/(mg/ kg)
CL (mL/ min/kg)
T1/2 (h)
Vdss (L/kg)
F (%)
Vercirnon 27
10 34
4.4 3.9
11 43
539 170
1.2/51 240/1100
99.1 98.4
6/147 5/49
0.56 0.65
33 26
1.3 1.2
0.83 1.1
10 38
stood out due to their superior potency in the buffer migration assay. It is also worth noting that several compounds showed a high LipE, which should correlate with drug-like properties and could warrant further development. Table 4 summarizes the representative pyrazine SAR. Due to synthetic feasibility considerations, we primarily focused on C3 substitution. 3-Methylaminopyrazine analog 25 was similarly CCR9 potent to the parent 3-aminopyrazine analog 14, while 3ethylamino analog 26 was significantly less potent. Interestingly, 3-hydroxyethylaminopyrazine 27 regained potency, possibly due to a favorable interaction of the terminal hydroxyl with the receptor. Further extending the chain length dramatically decreases potency (28). Both basic and acidic functional groups were not tolerated in this region (29, 30). With 3-ethanolamine as the optimal substitution, we briefly surveyed small substitutions on the C5 and C6 positions (31–33). Surprisingly, even small groups were not tolerated on these positions. This is contrary to the pyrimidine SAR where small substitutions are tolerated all around the ring. We reasoned that the binding conformation of 3-hydroxyethylaminepyrazines could be slightly different from those of aminopyrimidines. Compound 27 was selected for further in vivo mouse PK profiling.13 Table 5 details the head to head comparison of the in vitro/ in vivo profiles between 27 and Vercirnon. Though 27 is 3–4 less potent in calcium binding and buffer chemotaxis than Vercirnon, it is 3 more potent in the serum chemotaxis assay. We attributed the improved serum migration activity to the overall improved physiochemical properties which resulted in improved protein binding and significantly improved solubility. While displaying a similar mouse IV PK profile, 27 showed much improved oral absorption (38% vs 10% bioavailability). In addition, we did not observe any of the oral PK variability problems encountered in Vercirnon studies. The combined superior in vitro/in vivo profile should translate into better in vivo animal efficacy and clinical outcomes. In summary, we sought to improve both the chemical and physiochemical properties of Vercirnon by designing and synthesizing a series of heteroaryl-substituted biarylsulfonamides. The improved physiochemical properties indeed resulted in the identification of compounds with better in vitro and in vivo profiles. These compounds are candidates for further in vivo profiling, and the results from these studies will be reported in future.
References and notes 1. Powrie, F. Immunity 1995, 3, 171. 2. Powrie, F.; Read, S.; Mottet, C.; Uhlig, H.; Maloy, K. Novartis Found Symp. 2003, 252, 92. 3. Uhlig, H. H.; Powrie, F. Semin. Immunopathol. 2005, 27, 167. 4. Elson, C. O.; Cong, Y.; McCracken, V. J.; Dimmitt, R. A.; Lorenz, R. G.; Weaver, C. Immunol. Rev. 2005, 206, 260. 5. Naito, Y.; Takagi, T.; Yoshikawa, T. J. Gastroenterology 2007, 42, 787. 6. Strober, W.; Fuss, I. J. Gastroenterology 2011, 140, 1756. 7. Marsal, J.; Svensson, M.; Ericsson, A.; Iranpour, A. H.; Carramolino, L.; Marquez, G.; Agace, W. W. Eur. J. Immunol. 2002, 32, 3488. 8. Svensson, M.; Marsal, J.; Ericsson, A.; Carramolino, L.; Brodén, T.; Marquez, G.; Agace, W. W. J. Clin. Invest. 2002, 110, 1113. 9. Pabst, O.; Ohl, L.; Wendland, M.; Wurbel, M. A.; Kremmer, E.; Malissen, B.; Forster, R. J. Exp. Med. 2004, 199, 411. 10. Wendland, M.; Czeloth, N.; Mach, N.; Malissen, B.; Kremmer, E.; Pabst, O.; Forster, R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 6347. 11. (a) Keshav, S.; Vanasek, T.; Niv, Y.; Petryka, R.; Howaldt, S.; Bafutto, M.; Racz, I.; Hetzel, D.; Nielsen, O.; Vermeire, S.; Reinisch, W.; Karlen, P.; Schreiber, S.; Schall, T.; Bekker, P. PLoS One 2013, 8, e60094; (b) Walters, M. J.; Wang, Y.; Lai, N.; Baumgart, T.; Zhaom, B.; Dairaghi, D. J.; Bekker, P.; Ertl, L. S.; Penfold, M. E.; Jaen, J. C.; Keshav, S.; Wendt, E.; Pennell, A.; Ungashe, S.; Wei, Z.; Wright, J. J.; Schall, T. J. J. Pharmacol. Exp. Ther. 2010, 335, 65; (c) Chen, X.; Fan, J.; Fan, P.; Krasinski, A.; Li, L.; Lui, R.; McMahon, J.; Powers, J.; Zeng, Y.; Zhang, P. U.S. patent 20130225580A1.; (d) Arseneau, K.; Cominelli, F. Expert Opin. Invest. Drugs 2013, 22, 907; (e) Charvat, T.; Cheng, H.; Melikian, A.; Novack, A.; Pennell, A.; Powers, J.; Screenivas, P.; Sullivan, E.; Thomas, W.; Ungashe, S.; Zhang, P. PCT WO2009/038847A1.; (f) Anderson, E.; Biediger, R.; Chen, J.; Dupre, B.; Lory, P.; Market, R. V.; Monk, K. A.; Savage, M. M.; Tennyson, R.; Young, B. M. PCT WO2009/017719A2.; (g) Lehr, P.; Winiski, A. PCT WO2008/ 095908.; (h) Ungashe, S.; Wright, J.; Pennell, A.; Wei, Z.; Melikian, A. PCT WO2005/112925A1. 12. Calcium mobilization assay: Cells expressing CCR9 receptor were seeded in 384well plates. Ca2+ assays were conducted after overnight culture in the plates according to the manufacturer’s protocol using Screen QuestTM Fluo-8 no wash kit. Dye loading buffer was added to the cells and incubated for 45 min at 37 °C followed by 15 min incubation at room temperature. Compounds in the presence of 0.1% DMSO were applied to the cells during calcium flux measurement. Calcium flux was monitored for 90 s with compound application at 10th second. For antagonist mode, cells were preincubated with the compound at room temperature for 10 min before the application of the control agonist TECK at EC80 concentration of 0.01 lM obtained from doseresponse curves of control agonist. 13. Pharmacokinetics: mice (n of 3) were dosed both intravenously and orally with the test article. Plasma samples were collected at time points chosen to provide sufficient coverage of the absorption, distribution, metabolism and excretion phases of the test compound. LC–MS detected bioanalytical analysis was performed on the plasma samples utilizing a standard curve and quality control samples to provide enough precision and accuracy to determine the concentrations of test article for plasma from each time point. Noncompartmental pharmacokinetic analysis of the intravenous bioanalytical data provided area under the curve, clearance, volume of distribution, terminal half life parameters while similar analysis of plasma samples from animals dosed orally provided area under the curve, bio-available fraction and oral terminal half life pharmacokinetic parameters.
Acknowledgment We thank Dr. Hongwu Gao and the team at Chempartner for compounds synthesis, and Evotec for modeling supports.
Please cite this article in press as: Zhang, J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.046
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Biarylsulfonamide CCR9 inhibitors for inflammatory bowel disease Jing Zhang ⇑, Jan Romero, Audrey Chan, Jennifer Goss, Sabrina Stucka, Jason Cross, Brian Chamberlain, Mustafa Varoglu, Haoqun Chandonnet, Dominic Ryan, Blaise Lippa Cubist Pharmaceuticals, 65 Hayden Avenue, Lexington, MA 02421, United States
a r t i c l e
i n f o
Article history: Received 14 May 2015 Revised 10 June 2015 Accepted 12 June 2015 Available online xxxx
a b s t r a c t Inflammatory bowel disease, including Crohn’s disease and ulcerative colitis, affects millions of people worldwide. CCR9 has been shown to be a key chemokine receptor mediating the local inflammatory responses in the GI tract. The CCR9 inhibitor Vercirnon advanced to phase 3 clinical trials, but carries several liabilities which we sought to improve. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: IBD Crohn’s disease Ulcerative colitis CCR9 Sulfonamide
Inflammatory bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic inflammation in the gastrointestinal tract. It affects millions of people worldwide with >1 million new cases each year in the US alone. Despite extensive research, current therapeutic options for IBD are still very limited. Steroids are highly effective in achieving remission but long term use leads to significant side effects and drug tolerance. Immunosuppressants are often applied to maintain remission, but are sub-optimal in terms of efficacy and safety. In the last 10 years, the standard of care in the US has become antiTNF monoclonal antibodies (e.g. infliximab) in combination with the immunosuppressant azathioprine. About two thirds of the patients initially respond to this therapy, but only 50% remain in remission after a year of therapy. Thus safer and more effective therapeutic strategies for IBD are needed. Although the etiology of IBD has not been fully elucidated, genetic, immune and environmental risk factors have been implicated. Biopsy samples from patients with active disease show large numbers of leukocyte, lymphocyte and monocyte infiltrations in the intestinal/colonic mucosa. Data from a variety of immuneengineered IBD mouse models also suggest that chronic gastrointestinal tract inflammation results from a dysregulated immune response to otherwise harmless commensal flora.1–4 The adaptive immune system, particularly T cells, is believed to play a pivotal role in the induction and perpetuation of IBD pathogenesis.5,6
⇑ Corresponding author. Tel.: +1 781 640 2333. E-mail address: [email protected] (J. Zhang).
Chemokine receptors and their ligands regulate the migration and maturation of inflammatory and immune cells. CCR9 is a C– C chemokine receptor, expressed mainly in circulating lymphocytes. CCL25, the only identified ligand for CCR9, is primarily expressed in thymus and intestine which in turn results in enriched CCR9-positive cells in the intestine. Accumulating evidence indicates CCR9 expressing pro-inflammatory cells are involved in the IBD disease initiation and progression.7–10 Due to the high unmet medical need for IBD, and the strong biological validation of the CCR9/CCL25 pathway to mediate lymphocyte homing to the intestine, CCR9 has attracted a great deal of attention.11 Most notably, GSK/Chemocentryx was able to advance its CCR9 antagonist Vercirnon into a critical phase III clinical trial. Unfortunately, the clinical program was terminated in late 2013 due to insufficient efficacy in IBD. It is believed that the lack of clinical efficacy could have been due to Vercirnon’s poor physicochemical properties and inability to sufficiently inhibit CCR9 continuously. Most notably, the extremely poor solubility of Vercirnon has resulted in variable PO and SC PK in our laboratories. Structurally, it carries a biaryl ketone, which is a potential structural alert due to its reactivity. Herein we wish to report our efforts toward an improved CCR9 inhibitor based on the biarylsulfonamide scaffold (Fig. 1). Conformational analysis of Vercirnon shows that its ketone may form an intramolecular hydrogen bond with the sulfonamide NH, restricting the overall conformation of the biaryl ketone. The corresponding N–Me analog of Vercirnon abrogates the CCR9 binding affinity completely, supporting this conformational hypothesis, not but excluding other possibilities. Our strategy was to replace
http://dx.doi.org/10.1016/j.bmcl.2015.06.046 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhang, J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.06.046
2
J. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx O O S
Intramolecular HB O
NH
O
O S
N+
O-
NH
N
R
Cl
Vercirnon IC50 = 10 nM Chemotaxis (serum) = 539 nM logD = 3.7 LipE = 4.4 Solubility pH7.4/9.0 (ug/mL): 1.2/51
Cl
Figure 1. Vercirnon structure and design strategy.
O O S Cl
O O S NH
NH 2 Br +
a
Br
b
Cl O O S NH
B
O B
O
A
O O S NH
c
Cl
Ar
Cl
Cl
C
Scheme 1. General synthetic route. Reagents and conditions: (a) pyridine, 80 °C, (b) bis(pinacolato)diboron, Pd(dppf)2Cl2, KOAc, 1,4-dioxane, 100 °C, 50%; (c) ArBr, Pd(dppf)2Cl2, aq Na2CO3, 1,4-dioxane, 100 °C.
Table 2 SAR around the hetero-aryls
Table 1 Pyridine SAR
O O
O O S NH
1
S
6 5
N
NH
N
R
2
4 3
Cl
Cl
ID
R
CCR9 Ca2+ IC50 (nM)12
LipE
Chemotaxis (Buffer) IC50 (nM)
1 2 3 4 5 6 7 8 9
H 4-OMe 3-OMe 3-OH 3-NH2 3-CH2OH 3-CH2NH2 3-CH2OCH3 3-OH, Pyridine Noxide 3-OH, 4-CONH2 3-OH, 4-CH2OH
>10,000 2500 958 29 95 89 3163 3477 491
10000
2.9
—
29
2.9
1310
1000
0.3
—
55
3.2
—
29
3.2
23
26
3.8
23
N
1 N
4 OH N
12
N N
N
13 NH 2 N
the potentially problematic arylketone moiety with functionalized heterocycles, while retaining a nitrogen atom in the 20 -position to maintain the critical intramolecular hydrogen bonding motif. It was envisaged that suitably positioned solubilizing groups extending from the heterocycle would improve the overall physiochemical properties. The analog synthesis was straightforward as depicted in Scheme 1. We first explored 2-pyridyl as the biarylketone replacement (Table 1). Although the parent 2-pyridyl analog 1 was inactive toward CCR9, preliminary substituted pyridine SAR studies identified 3-methoxy-2-pyridyl analog 3 with 1 lM activity.
N
14
NH 2
N
N
15 NH 2
Interestingly, simply removing the methyl off the methoxy gave compound 4 with 29 nM CCR9 inhibition. Further analoging on the C3 position revealed that only small functional groups were
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J. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx Table 3 Pyrimidines SAR
Table 4 Pyrazines SAR O O S
NH
3 N 4
O O
2
N 5
R
S
1 6
2
CCR9 Ca2+ IC50 (nM)
R
N
Ca2+ LipE
Chemotaxis (Buffer) IC50 (nM)
ID
3.2
—
14
N
N
4.5
750
Chemotaxis (Buffer) IC50 (nM)
29
3.2
23
N
25
3.3
235
N
421
1.6
—
34
3.9
34
614
2.8
—
3604
2.5
—
812
5.9
—
268
2.7
—
1005
2.0
—
715
1.7
—
N
25
OH
HN
NH 2
N
N
4
5.0
30
26 HN
OH
N
NHMe
18
Ca2+ LipE
N
11
N
4
NH 2
16
17
3
CCR9 Ca2+ IC50 (nM)
R
NH 2
N
5
N
N
55
13 N
6R
Cl
Cl
ID
NH 1 N
N
27
N
27
4.0
HN
—
OH
N
OH
OH
HN
19
N
N
HN
214
3.9
14
NH 2
4.6
HN
N
N
30 HN
N
21
9
N
3.7
COOH
N
105
OH
N
31 HN
N
22
26
N NH2
3.9
N
32 11
5.6
HN
325
OH
Cl
OH N
N
6
24
H N
N
O OH
OH
190
N
23 N
N
N
340
OH
N
N
29
N
20
N
OH
N
—
OH N
N
28
3.7
60
OH
tolerated, and a hydrogen bond donor was critical for enzymatic activity. While 3-amino and 3-hydroxymethyl substituents were well tolerated (potency for 5 and 6 is within 3 of 3-hydroxyl analog 4), moderately basic functional groups such as 3-aminomethyl analog 7 was not tolerated. For initial SAR studies around the pyridine ring, we chose the 3-hydroxy analog as our next benchmark. Small substituents on pyridine positions 5 and 6 were tolerated, while position 4 could accommodate a variety of functional groups. We sought to improve the physicochemical properties via C4 substitutions. The 4-carboxylamide and 4-hydroxymethyl analogs 10 and 11 stood out as two of the best. Analog 11 had an enzymatic IC50 of 8.7 nM and a functional IC50 of 330 nM in the buffer migration assay. To further improve physiochemical properties and CCR9 potency, we briefly surveyed some other nitrogen-containing heterocycles (Table 2). Interestingly, 2-pyrimidine analog 12 improved
33
N HN
OH
the activity over the parent 2-pyridine by >10. The aminopyrimidine, aminopyrazine and aminopyridazine analogs 13–15 all displayed similar enzymatic potencies to the lead 3hydroxylpyridine analog 4. However, the aminopyrazine and aminopyridazine analogs 14 and 15 showed 50 improvement in CCR9 functional activity over 4. Due to synthetic feasibility and metabolic stability considerations, we decided to focus on the pyrimidine and pyrazine scaffolds for further SAR studies. Representative pyrimidine SAR is detailed in Table 3. As in the pyridine series, o-hydroxyl 16 gave better CCR9 inhibition than o-amino substituent. Small, polar substituents at the C2 position gave the best activity (17, 4 nM). As the size increases, the activity gradually deteriorates (18, 19). The C6 position tolerates a variety of functional groups in terms of size and polarity (21–24). Among them, 6-dimethylamino and 6-ethyl analogs 21 and 24
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J. Zhang et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 5 In vitro/in vivo (mice) profiles of 27 versus Vercirnon Compd
Ca2+ IC50 (nM)
LipE
Chemotaxis (Buffer) IC50 (nM)
Chemotaxis (Serum) IC50 (nM)
Sol. pH 7.4/ 9 (lg/mL)
hPPB (% Bound)
HLM/MLM CL (ml/min/kg)
DN_AUC IV (lg hr/mL)/(mg/ kg)
CL (mL/ min/kg)
T1/2 (h)
Vdss (L/kg)
F (%)
Vercirnon 27
10 34
4.4 3.9
11 43
539 170
1.2/51 240/1100
99.1 98.4
6/147 5/49
0.56 0.65
33 26
1.3 1.2
0.83 1.1
10 38
stood out due to their superior potency in the buffer migration assay. It is also worth noting that several compounds showed a high LipE, which should correlate with drug-like properties and could warrant further development. Table 4 summarizes the representative pyrazine SAR. Due to synthetic feasibility considerations, we primarily focused on C3 substitution. 3-Methylaminopyrazine analog 25 was similarly CCR9 potent to the parent 3-aminopyrazine analog 14, while 3ethylamino analog 26 was significantly less potent. Interestingly, 3-hydroxyethylaminopyrazine 27 regained potency, possibly due to a favorable interaction of the terminal hydroxyl with the receptor. Further extending the chain length dramatically decreases potency (28). Both basic and acidic functional groups were not tolerated in this region (29, 30). With 3-ethanolamine as the optimal substitution, we briefly surveyed small substitutions on the C5 and C6 positions (31–33). Surprisingly, even small groups were not tolerated on these positions. This is contrary to the pyrimidine SAR where small substitutions are tolerated all around the ring. We reasoned that the binding conformation of 3-hydroxyethylaminepyrazines could be slightly different from those of aminopyrimidines. Compound 27 was selected for further in vivo mouse PK profiling.13 Table 5 details the head to head comparison of the in vitro/ in vivo profiles between 27 and Vercirnon. Though 27 is 3–4 less potent in calcium binding and buffer chemotaxis than Vercirnon, it is 3 more potent in the serum chemotaxis assay. We attributed the improved serum migration activity to the overall improved physiochemical properties which resulted in improved protein binding and significantly improved solubility. While displaying a similar mouse IV PK profile, 27 showed much improved oral absorption (38% vs 10% bioavailability). In addition, we did not observe any of the oral PK variability problems encountered in Vercirnon studies. The combined superior in vitro/in vivo profile should translate into better in vivo animal efficacy and clinical outcomes. In summary, we sought to improve both the chemical and physiochemical properties of Vercirnon by designing and synthesizing a series of heteroaryl-substituted biarylsulfonamides. The improved physiochemical properties indeed resulted in the identification of compounds with better in vitro and in vivo profiles. These compounds are candidates for further in vivo profiling, and the results from these studies will be reported in future.
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Acknowledgment We thank Dr. Hongwu Gao and the team at Chempartner for compounds synthesis, and Evotec for modeling supports.
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