Novel, Broad-Spectrum Anticonvulsants Containing a Sulfamide

Apr 23, 2009 - Phone: 215-628-5530. Fax: 215-540-4612. E-mail: [email protected]., †. These authors contributed equally to this work. This articl...
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7528 J. Med. Chem. 2009, 52, 7528–7536 DOI: 10.1021/jm801432r

Novel, Broad-Spectrum Anticonvulsants Containing a Sulfamide Group: Advancement of N-((Benzo[b]thien-3-yl)methyl)sulfamide (JNJ-26990990) into Human Clinical Studies )

Michael H. Parker,§,† Virginia L. Smith-Swintosky,§,† David F. McComsey,§ Yifang Huang,§ Douglas Brenneman,§ Brian Klein,§ Ewa Malatynska,§ H. Steve White, Michael E. Milewski,§ Mark Herb,§ Michael F. A. Finley,§ Yi Liu,§ Mary Lou Lubin,§ Ning Qin,§ Robert Iannucci,‡ Laurent Leclercq,^ Filip Cuyckens,^ Allen B. Reitz,§ and Bruce E. Maryanoff*,§ §

Research and Early Development, Johnson & Johnson Pharmaceutical Research & Development, Spring House, Pennsylvania 19477-0776, Drug Metabolism and Pharmacokinetics, Johnson & Johnson Pharmaceutical Research & Development, Raritan, New Jersey 08869-0602, ^ Drug Metabolism and Pharmacokinetics, Johnson & Johnson Pharmaceutical Research & Development, 2340 Beerse, Belgium, and Department of Pharmacology & Toxicology, College of Pharmacy, University of Utah Health Sciences Center, Salt Lake City, Utah 84112-5820. † These authors contributed equally to this work.

)

‡

Received November 13, 2008

In seeking broad-spectrum anticonvulsants to treat epilepsy and other neurological disorders, we synthesized and tested a group of sulfamide derivatives (4a-k, 5), which led to the clinical development of 4a (JNJ-26990990). This compound exhibited excellent anticonvulsant activity in rodents against audiogenic, electrically induced, and chemically induced seizures, with very weak inhibition of human carbonic anhydrase-II (IC50 = 110 μM). The pharmacological profile for 4a supports its potential in the treatment of multiple forms of epilepsy, including pharmacoresistant variants. Mechanistically, 4a inhibited voltage-gated Naþ channels and N-type Ca2þ channels but was not effective as a Kþ channel opener. The pharmacokinetics and metabolic properties of 4a are discussed.

Topiramate (1)1 is an anticonvulsant drug that is marketed worldwide for the treatment of epilepsy and migraine (Chart 1).2 Besides these approved indications, other CNS therapeutic uses have been reported and discussed, such as the treatment of eating disorders, nerve injury, alcohol and drug dependence, neuropathies, restless legs syndrome, essential tremor, bipolar disorder, and schizophrenia.3 This broad dimensionality is thought to be derived from topiramate’s multiple mechanisms of neuronal modulation, which serve to expand its clinical pharmacology.4 Thus, the terms “neurostabilizer” and “neurostabilizing agent” have emerged to characterize this type of control of neuronal excitability.4a,5 We have been interested for some time in discovering follow-up compounds to 1. In the early 1990s, we identified topiramate analogue 2 (RWJ-37947), which exhibited 8-10 times greater potency than 1 in the rat maximal electroshock seizure (MESa) test and a 2-fold longer plasma half-life in rats (Chart 1).6 However, 2 was not advanced into preclinical development because of its high potency in inhibiting carbonic anhydrase (CA) enzymes,6 which can lead to undesirable side effects in certain patients. Following observations with 1 relating to weight loss and glycemic control in patients and animals,7 we came to investigate related compounds as *To whom correspondence should be addressed. Phone: 215-6285530. Fax: 215-540-4612. E-mail: [email protected]. a Abbreviations: AUC, area under the curve (total drug exposure); Bic, bicuculline; CA, carbonic anhydrase; CL, clearance from plasma; EGTA, ethylene glycol bis(β-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid; GABA, γ-aminobutyric acid; KA, kainic acid; MES, maximal electroshock seizure; NMDA, N-methyl-D-aspartic acid; Pic, picrotoxin; PTZ, pentylenetetrazol; TD50, dose that is toxic in 50% of the test subjects; Vdss, volume of distribution at steady-state.

pubs.acs.org/jmc

Published on Web 04/23/2009

potential antidiabetic/antiobesity agents. The direct sulfamide isostere of topiramate, 3 (RWJ-37082), was found to be essentially devoid of anticonvulsant activity6 and to exhibit just very weak inhibition of carbonic anhydrase-II (CA-II) (Chart 1).8 We pursued the synthesis of other sulfamide compounds with simplified structures (4a-k, 5) for phenotypic assessment in the metabolic arena but also continued to screen for anticonvulsant activity. Consequently, we were able to identify some novel sulfamide derivatives with promising anticonvulsant properties that differ in structure from recently reported anticonvulsant sulfamides.9,10 Herein, results on our series of sulfamides are presented with a particular emphasis on 4a (JNJ-26990990, Chart 1), which was taken into human clinical trials. Results and Discussion For assessing newly prepared sulfamides, we established a distinct biological testing protocol. New compounds were first examined for the virtual absence of CA-II inhibition. Compounds that passed this CA screen (IC50 > 10 μM; CO2 hydration assay) were tested for CNS behavioral effects in mice and for anticonvulsant activity in the mouse MES test. The key point was to discover novel broad-spectrum anticonvulsants with suitable characteristics for eventual clinical development. A compound of interest would possess a pharmacological profile extending beyond that of currently known antiepileptic drugs, including 1. Work on the synthesis and testing of several sulfamide derivatives (Table 1) led to the selection of anticonvulsant 4a for human clinical development. Chemical Synthesis. Some synthetic routes to sulfamides were described in our previous papers in relation to exploring r 2009 American Chemical Society

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Chart 1. Sulfamates and Sulfamides

Table 1. Sulfamide Compounds and Biological Testing Data

Table 2. Mouse MES ED50 Data for Selected Compoundsa compd

compd

G

4a 4b

H H

4c 4d 4e

2-Me H H

4f

5-Cl

4g 4h 4i 4j 4k 5f 1

5-F 4-Br 5-Br 5-Me H

Z

MES, po,a t (h), effect CA-II inh,b IC50 (μM)

S O

0.5, 1/5; 2, 3/5 0.5, 0/5; 2, 0/5 0.5, 5/5; 2, 0/5d O 0.5, 4/5; 2, 0/5 NH 0.5, 1/5; 2, 0/5 NMe 0.5, 0/5; 2, 0/5 0.5, 5/5; 2, 0/5d S 0.5, 0/5; 2, 0/5 0.5, 0/5; 2, 3/5d S 0.5, 0/5; 2, 4/5 S 0.5, 0/3; 2, 1/3 S 0.5, 0/3; 2, 1/3 S 0.5, 0/3; 2, 2/3 CHdCH 0.5, 2/5; 2, 1/5 0.5, 1/5; 2, 0/5

110,c 66 7.1 25 300c 48

ED50, po (mg/kg) c

119 >400 345 231 >400 123 43.8e

4a 4b 4e 4g 4j 4k 1

95% CI

neurotoxb

112-126

TD50 = 182 mg/kgd 0/8 (400) 1/8 (400) 2/8 (300) 3/8 (400) 3/8 (150)

304-390 165-299 109-137

a

Maximal electroshock seizure model at 1 h post dosing, performed at NeuroAdjuvants. CI, confidence interval. b Rotorod neurotoxicity in mice at the ip dose in mg/kg given in parentheses. The effect is presented as the number of animals responding out of the total number of animals tested. c Performed in house; result at 3 h postdosing (time of peak effect). d Oral TD50 > 500 mg/kg in rats (see text). e Taken from ref 6 (ED50 = 47.6 mg/kg at 2 h; ED50 = 53.5 mg/kg at 4 h).

8.4c 30 3.7 ∼100c,e 45c 21 130 2.1g

a Oral dose of 100 mg/kg, unless otherwise noted. The result is presented as the number of animals responding out of the total number of animals tested. b IC50 values for the inhibition of human CA-II were determined by using a CO2 hydration assay either in house (refs 8a-8d) or by Cerep, an independent research laboratory (refs 14 and 15). c IC50 value determined in house. d Oral dose of 300 mg/kg. e 44% inhibition at 100 μM. f Racemate. g Taken from ref 8a (CO2 hydration assay).

Scheme 1. Synthesis of 4a

the inhibition of carbonic anhydrases.8 Compounds 4a-f and 4h-k were readily prepared by reductive amidation of the requisite aldehyde precursor with sulfamide and NaBH4. The synthesis of 4a from aldehyde I is illustrated in Scheme 1. Compound 4g was prepared as follows: the methyl group of 5-fluoro-3-methylbenzothiophene was brominated; an azide displacement was performed, and the azide was reduced to the amine with triphenylphosphine; then the primary amine was heated with sulfamide. Compound 5 was obtained by reductive amination of 3-acetylbenzothiophene with formic acid and formamide, followed by hydrolysis; then the primary amine was heated with sulfamide. Anticonvulsant Screening. Sulfamide derivatives were assessed initially for a lack of inhibition of CA-II8 (IC50>30 μM) and for activity in the mouse MES test (Table 1).1a,12 The MES assay entails the application of an electrical current to

induce tonic extension of the hind limbs, which will be inhibited by an anticonvulsant compound. The results are reported according to the number of responders out of the total number of animals per group, at 0.5 and 2 h. Compounds were tested by oral administration. The mouse MES anticonvulsant activity for sulfamide compounds can be compared for oral dosing at the 2 h time point because of the delay in uptake by this route (Table 1). Among compounds 4a-k and 5, benzothiophenes 4a, 4f (5-Cl), 4g (5-F), and 4j (5-Me) showed reasonable activity. Mouse MES ED50 data were obtained on certain sulfamides to establish a more robust comparison (Table 2). Compounds 4a and 4k were relatively close in potency (∼120 mg/kg); 4e and 4g were weaker (230-350 mg/kg), and 4b and 4j did not show meaningful anticonvulsant activity (>400 mg/kg). Compounds 4b, 4e, 4g, 4j, and 4k were subjected to a mouse rotorod neurotoxicity screen (Table 2), and the ED50 values for 4e and 4g appeared to be much lower than doses that produce substantial neurotoxicity. Compound 4a was only assessed in a rat neurotoxicity screen (vide infra). Relative to anticonvulsants that contain a sulfamide functionality, it should be noted that there have been two recent reports on this subject.9 Most of the reported compounds possess a sulfamide group with N,N-/N,N0 -disubstitution or N,N,N0 -trisubstitution. However, two primary sulfamides, PhCH2NHSO2NH2 (6) and BuNHSO2NH2 (7), are of particular interest from the perspective of our compound series. Benzyl derivative 6 was fully active (3/3 animals) in the mouse MES test at 0.5 h at 100 and 300 mg/kg and at 4 h at 300 mg/kg, with an ED50 of 440 mg/kg at 0.5 h (time of peak effect).9b By contrast, butyl derivative 7 was inactive in the MES test.9b Inhibition of Carbonic Anhydrase-II. Various sulfamides were examined for inhibition of human CA-II by using a CO2 hydration assay (Table 1), which we have described previously.8a-8d The inhibition of CA-II was relatively weak,

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Chart 2. Structures for Reference Anticonvulsants

with IC50 values ranging from 4 to 300 μM. For comparison, sulfamate 1 had a CA-II IC50 value of 2.1 μM8a (and Ki values of 0.3-0.6 μM8a,8b). The weak CA-II inhibition among this collection of sulfamides is consistent with our prior reports on the behavior of other sulfamides with diverse structures.8 By use of a CO2 hydration assay (at 05 C),8a-8d 4a was found to be a very weak inhibitor of CAII, with an IC50 value of 110 μM, as well as a very weak inhibitor of CA-I (IC50 = 25 μM). Independent assessment of 4a by an external laboratory, Cerep,13 involving human erythrocyte CA-II and hydration of carbon dioxide at 22 C with fluorimetric detection14 yielded an IC50 value of 66 μM,15 which is reasonably consistent with the value determined in our laboratory. Advanced Pharmacological Assessment of 4a. We selected 4a for detailed evaluation as an anticonvulsant to establish its pharmacological profile.16 The principal reasons for its selection were (1) a substantial absence of CA-II inhibition, (2) reasonable oral potency in the mouse MES test, (3) low neurotoxicity in rats, and (4) favorable pharmacokinetics in rats and dogs, with high oral bioavailability and long duration (vide infra). Sulfamide 4a was found to exhibit broadspectrum anticonvulsant activity in rodents against electrically induced and chemically induced seizures. The oral ED50 values for 4a in the mouse and rat MES tests at the time of peak effect were 119 mg/kg, po (3 h) and 34 mg/kg, po (2 h), respectively. MES ED50 values for 1 were 47.6 mg/kg (2 h) and 53.5 mg/kg (4 h) in mice and 6.6 mg/kg in rats.6 Compound 4a effectively blocked forelimb clonic seizures in mice that were induced by subcutaneously administered bicuculline (Bic), picrotoxin (Pic), or pentylenetetrazol (PTZ), with ED50 values (0.25 h) of 156, 106, or 161 mg/ kg, ip, respectively. This potency level for chemically induced seizures is better than that for 1 (ED50>500 mg/kg), valproic acid (ED50>200 mg/kg), and levetiracetam (ED50>540 mg/ kg) (Chart 2). The intravenous PTZ seizure-threshold test provides a sensitive parametric method for assessing seizure threshold.16 In this test, a compound does not have to completely prevent the occurrence of a seizure but only delay its appearance, indicating its ability to modify seizure threshold. The iv PTZ test can be used to determine whether a compound is able to increase (anticonvulsant) or decrease (proconvulsant) seizure threshold. Two doses of the compound were employed, the first corresponding to the ED50 for protection against MES seizures and the second corresponding to the median toxic dose (TD50), which is the dose that produces rotorod impairment in 50% of the mice

tested.17 Nine mice per dose group were injected ip with vehicle (0.5% aqueous methylcellulose) or a solution of 4a. Fifteen minutes postdosing, a saline solution of PTZ was infused into the tail vein of the mice at a constant rate. The times from the start of the infusion to the appearance of the first twitch and the onset of clonus were recorded. An increase in the mg/kg dose of PTZ to produce a first twitch or clonic seizure suggests that the test compound increases seizure threshold (anticonvulsant). Compared to vehicletreated mice, 4a at its ED50 (107 mg/kg, ip) and TD50 (182 mg/kg, ip) doses markedly increased the seizure threshold for twitch and clonus (at ED50, 129% and 147%; at TD50, 141% and 159%). In comparison, 1 had no effect on seizure threshold at its ED50 and was proconvulsant at its TD50. Reported sulfamides 6 and 7 (vide supra) were inactive in blocking PTZ-induced seizures.9b Audiogenic seizures in mice, induced by sound, are characterized by wild running followed by a loss of righting reflex with forelimb and hindlimb tonic extension. Mice that do not display hindlimb tonic extension on drug treatment are considered protected.17 Eight mice per dose, and at least four doses, were used to establish an ED50 value (at time to peak effect). Compound 4a, administered ip 1 h prior to sound induction, was effective in this model with an ED50 value of 21 mg/kg (5-fold less potent than 1; ED50 = 4.2 mg/kg). The kindling model is a useful adjunct to traditional anticonvulsant tests for identifying an anticonvulsant compound’s potential utility for treating pharmacoresistant limbic epilepsy, which is manifested in complex partial seizures with secondarily generalized seizures. Kindled seizures also provide a model of focal seizures, which facilitates the study of the complex brain networks that contribute to seizure spread and generalization from a focus.17 In studies conducted via the NINDS unit of the NIH, the rat hippocampal kindling model was used.18 Efficacy (ED50 value) was measured as the ability of a compound to modify the seizure score (severity of spread) and after-discharge duration (excitability) of the generalized seizures. With this approach, a test compound that reduces the seizure score from 5 to 3, without affecting the after-discharge duration, would be useful presumably against secondarily generalized seizures. In contrast, a compound that reduces the seizure score from 5 to less than 1, while reducing the after-discharge duration, would be anticipated to be effective against focal seizures. Compound 4a (in 0.5% aqueous methylcellulose) exhibited very good anticonvulsant activity in this model with an ED50 of 38.9 mg/kg (peak activity at 15 min, sustained at 1 h). Seizure scores were significantly reduced from 5 to 0 in six

Article

Figure 1. Frequency distribution analysis for seizures in individual rats (N = 8 animals) in the hippocampal kindling model after treatment with various anticonvulsants (4a, 1, ethosuximide, phenytoin, carbamazepine, and valproic acid; vehicle, 0.5% aqueous methylcellulose). The color code represents the number of rats out of eight that evinced the particular seizure response noted (seizure free, focal seizure, global seizure). Seizure scores were assessed at the time of peak effect and at the maximal effective dose.

out of eight rats (p = 0.0001) (viz. Figure 1), with a mean seizure score of (1.6 ( 0.7)/(0.9 ( 0.6) and statistically significant reduction of the after-discharge duration (64%; p26 mg/kg; similar protection was observed with valproic acid but only at neurotoxic doses (>300 mg/kg) (Chart 2). With 1, only one out of eight treated rats showed complete protection at maximally effective doses. Basically, topiramate and ethosuximide were ineffective in this model, whereas phenytoin, carbamazepine, and valproic acid significantly suppressed seizure activity (Chart 2) but at dose levels associated with neurotoxicity (Figure 1). Thus, in this antiepileptic model 4a exhibited superior activity, much better than that of various marketed antiepileptic drugs. Acute neurotoxicity for 4a was characterized in rats by the NINDS anticonvulsant screening process.17 Abnormal neurological status was determined by three tests: the positional sense test, muscle tone test, and the gait-and-stance test. The inability of a rat to perform normally in at least two of these tests indicates that the animal has some neurological deficit. The positional sense test evaluates the rat’s ability to quickly lift its leg back to a normal position after it has been gently lowered over the edge of a table. A neurological deficit is indicated by the inability to do this task. The gait-and-stance task measures the ability of the rat to maintain normal gait and posture. A neurological deficit is indicated by a circular or zigzag gait, ataxia, abnormal spread of the legs, abnormal body posture, tremor, hyperactivity, somnolence, stupor, or catalepsy. Finally, normal animals have a certain amount of skeletal muscle tone that on handling is apparent to the experimenter, and a neurological deficit is indicated by a loss of such tone, as characterized by hypotonia or flaccidity. Compound 4a, at doses up to 500 mg/kg, po (in 0.5% aqueous methylcellulose), showed no adverse effects, resulting in a significant safety margin between behavioral efficacy (rat oral MES ED50) and neurotoxicity (rat TD50) for a protective index (rat oral TD50/MES ED50) of >15-fold. The oral protective index for 1 in rats is 25.

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Mechanistic Studies with 4a. Many antiepileptic drugs have multiple mechanisms of action, often involving effects on different ion channels. For example, various active agents target voltage-gated sodium and calcium ion channels. Anticonvulsant drugs, such as phenytoin, lamotrigine, and 1, are known to inhibit voltage-gated Naþ channels (Chart 2).19 Additionally, in human epilepsy there are increased levels of brain Naþ channel mRNA.20 Given these findings, we probed whether 4a had any effects on Naþ channel function. Whole-cell patch-clamp recordings in CHL1610 cells stably transfected with rat Nav1.2, a voltage-gated Naþ channel highly expressed in the brain (e.g., hippocampus), demonstrated that 4a causes a dose-dependent, voltage-dependent blockade of these channels, with virtually no inhibition at -107 mV and an IC50 =48 μM at -67 mV (N=3). Similar Naþ channel blockade was observed for phenytoin, lamotrigine, and 1 (IC50 = 22, 35, and 97 μM, respectively). By contrast, gabapentin (Chart 2) had no effect on Naþ channel activity in this assay up to a concentration of 300 μM. These results suggest that the inhibitory effects of 4a on voltagegated Naþ channels in vitro may contribute to its anticonvulsant activity in vivo. Lamotrigine, felbamate, levetiracetam, and 1 inhibit high voltage-activated calcium channels, which may contribute to their pharmacological activity in epilepsy, neuropathic pain, and migraine (Chart 2).21 Compound 4a was studied in the neuronal (N-type) subgroup of the voltage-activated calcium channels. Two different in vitro assays that utilize the same heterologous cell line expressing the R, β, and R2-δ subunits, comprising the functional channel, were used to test for rat N-type calcium channel activity. First, 4a was found to inhibit this activity in a concentration-dependent manner (IC50 =28 μM) by using a fluorescence-based assay to measure calcium influx in response to depolarization. Second, whole-cell patch-clamp electrophysiology was employed to directly assess rat N-type channel activity in the absence and presence of 4a. With low frequency stimulation, we observed concentration-dependent increases in inhibition for 4a (IC50 =133 μM; 0.07 Hz) without a change in the currentvoltage relationship of activation (data not shown). Furthermore, 4a at 30 μM demonstrated an increase in inhibition with 5-Hz stimulation compared with that at 0.07-Hz stimulation: 30 ( 2% for 5 Hz vs 20 ( 0.5% for 0.07 Hz (N=3). These in vitro data suggest that 4a possesses moderate usedependent N-type calcium channel blocking activity. The M-current plays a role in regulating the resting membrane potential of neurons and thereby modulates neuronal activity, and a key component of the M-current in native cells is the voltage-gated potassium channel, known as KCNQ2 or Kv7.2.22 Human mutations in the gene that encodes KCNQ2 lead to a form of epilepsy, and the anticonvulsant retigabine is a potent KCNQ2-channel opener.23 Compound 4a was examined at 300 μM for its effect on whole-cell patch-clamp recordings of cells stably transfected with rat KCNQ2 by using an M-current protocol.16 At membrane potentials between -70 and -30 mV, 4a was very weak in increasing KCNQ2 current, indicating a lack of KCNQ2 channel opening activity. Whole-cell patch-clamp studies in cultured primary mouse cortical neurons (performed at the NINDS) indicated that 4a (100 μM at -70 mV) has weak (but statistically significant) effects on excitatory and inhibitory amino acid receptor currents for N-methyl-D-aspartic acid (NMDA) (23% inhibition of the current evoked by 1 μM glycine and 10 μM

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Chart 3. Structures of Metabolites from 4a

NMDA), kainic acid (KA) (8% inhibition of the current evoked by 100 μM KA), and γ-aminobutyric acid (GABA) (2% increase in the current evoked by 5 μM GABA).16 Compound 4a was profiled in binding assays that were conducted by the contract laboratory Cerep at a concentration of 10 μM.14 Thus, 4a was found to have virtually no affinity for GABA and central benzodiazepine receptors, as well as for chloride ion channels. Pharmacokinetics and Metabolism of 4a.24 Early on, sulfamide 4a (2 μM) was evaluated for metabolic stability in vitro by using mouse, rat, and human liver microsomes. After a 10 min incubation period in the microsomal preparations, the percentages remaining were 83% in mouse, 76% in rat, and 90% in human, which suggests reasonably high stability to hepatic transformation. The half-life of 4a in a standard human liver microsomal preparation was >100 min. The in vitro metabolism of 4a (10 μM) was studied in cryopreserved rat, dog, and human hepatocytes. In contrast, 4a was extensively metabolized in these cryopreserved hepatocytes mainly to benzothiophene S-oxide M1 (Chart 3, MW=258). The identity of M1 was initially established by mass spectrometry and later confirmed by independent synthesis (vide infra). Favorable pharmacokinetic results were obtained with 4a in mice, rats, dogs, and monkeys. Data from single-dose oral and iv administration of 4a to mice and rats are discussed herein. In mice, oral doses of 4a at 30 mg/kg were rapidly absorbed and eliminated; after iv administration of a 3 mg/kg bolus dose, 4a was eliminated rapidly. Volume of distribution at steady state (Vdss) greatly exceeded the volume of total body water (725 mL/kg), indicating extensive distribution outside the plasma compartment. The following parameters were obtained in male mice: F, 59%; oral Cmax, 3.9 μg/L; oral t1/2, 1.5 h; iv t1/2, 1.5 h; Vdss, 2.9 L/kg; CL, 1.8 L/(h 3 kg). In rats, 4a was administered at oral doses of 2 or 10 mg/kg to adult males. After both doses, absorption and elimination were rapid. On the basis of mean Cmax and AUC values, there were dose-related increases in exposure. At 2 and 10 mg/kg, the mean values of F were 89% and 96%, respectively. After iv administration at 2 mg/kg, 4a was eliminated rapidly and the Vdss values greatly exceeded the volume of total body water in the rat (670 mL/kg), suggesting extensive distribution of 4a outside plasma. Thus, after oral administration, 4a was highly bioavailable; it was absorbed rapidly and eliminated at a moderate rate. Plasma and brain concentrations of 4a were measured in male rats (N=3) after a dosing at 30 mg/kg, po (0.5% aqueous methylcellulose). After precipitation of proteins from the samples with acetonitrile, the resulting supernates were analyzed by LC/MS. Peak plasma and brain levels occurred at 30 min postdosing and decreased gradually over time in parallel, with a 10-fold decrease from peak at 6 h. Brain levels were ∼2.5-fold higher than plasma levels throughout the 6-h period. At 2 h postdosing, the concentrations in plasma and brain were 19 ( 6 and 47 ( 15 μM, respectively.

Plasma and urine samples from rats were used to identify in vivo metabolites, which are based on molecular weight and fragmentation data from MS/MS (electrospray) analysis and independent synthesis in the case of major metabolite M1. An authentic sample of M1 was readily produced by reacting 4a with m-chloroperbenzoic acid. In analyzing 4 h plasmas from male rats that were dosed orally (25 mg, 0.5% Methocel suspension) with radiolabeled 4a22 (14C-label located at the CH2 group of 4a; 59.2 kBq/mg), we found that M1 and benzothiophene-3-carboxylic acid, M4, accounted for 31% and 9% of total radioactivity, respectively, while 60% of 4a was unchanged. In urine, M1 represented 4877% of the administered dose over a 24 h period. Other metabolites that were detected in urine included M4 (∼0.5% in 8-24 h) and its glycine conjugate M5 as a secondary metabolite (4.2-8.6% in 8-24 h) (Chart 3). Minor metabolites M2 and M3, which correspond to N-acetyl 4a and an O-glucuronide of a monooxygenated metabolite, respectively, were detected by mass spectrometry. The major biotransformation products in human urine and plasma were derived from the same pathways. In plasma, M1 accounted for 78-100% of the drug-related compounds in the 500-mg dose group, up to 72 h. In urine, M1 accounted for 46-75% of the drug-related compounds in the 500 mg dose group, up to 24 h. This major transformation of 4a to M1 in humans is consistent with the in vivo observation of major M1 formation in rats. M1 was lower than 4a in the plasma of rats because of rapid urinary excretion. Conclusion Epilepsy is a chronic neurological condition that affects at least 50 million people worldwide, including approximately three million Americans. Although effective anticonvulsant drugs for the treatment of epilepsy have been available since the early 1900s, unmet medical needs remain. Approximately 15% of epileptics are not adequately treated, and another 20% endure intractable seizures. Next-generation anticonvulsants should have broad-spectrum anticonvulsant activity against multiple seizure types, accompanied by good safety and tolerability, as well as improved efficacy against refractory epilepsy. Anticonvulsant drugs have been effective in treating other neurological disorders besides epilepsy, such as neuropathic pain, bipolar disorder/depression, migraine, and substance abuse. Compound 4a exhibited broad-spectrum anticonvulsant activity in rodents against audiogenic, electrically induced, and chemically induced seizures, with very weak inhibition of human CA-II (IC50=110 μM). A summary of in vivo and in vitro pharmacological results for 4a are presented in Table 3. The very weak CA-II inhibition signifies that this mechanism is not likely to be a meaningful source of its anticonvulsant action. This point is important relative to our perspective that CA-II inhibition is not a major contributor to the anticonvulsant pharmacology of topiramate.8a,8b By limiting seizure

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Table 3. Summary of Testing Results for 4a test mouse MES, po, 3 h rat MES, po, 2 h mouse scBic, ip mouse scPic, ip mouse scPTZ, ip mouse ivPTZ, ipb mouse aud seiz, ip, 1 hc rat hipp kindl, ip, 1 hd mouse TD50, ip rat TD50, po in vitro rat Nav 1.2e in vitro rat Cav 2.2f in vitro rat Kv 7.2g inhibition of CA-II

4a result

ref cmpd

ED50 = 119 mg/kg ED50 = 34 mg/kg ED50 = 156 mg/kg ED50 = 106 mg/kg ED50 = 161 mg/kg twitch: 129% incr clonus: 147% incr 21 mg/kg 38.9 mg/kg 182 mg/kg >500 mg/kg IC50 = 48 μM (-67 mV) IC50 = 28 μM very weak effect IC50 = 110 μM

1 1 1 1 1 1

ref result

1 1

ED50 = 47.6 mg/kga ED50 = 6.6 mg/kg ED50 > 500 mg/kg ED50 > 500 mg/kg ED50 > 500 mg/kg twitch: 0% incr clonus: 0% incr 4.2 mg/kg no effect

1

IC50 = 97 μM (-67 mV)

1

IC50 = 2.1 μM

a

Result is for a 2-h time point; ED50 = 53.5 mg/kg at 4 h (ref 6). b Increase (incr) in seizure threshold for twitch and clonus response at the ED50 of 4a (107 mg/kg). c Blockade of audiogenic seizures. d Reduction of seizure scores in the rat hippocampal kindling model. e Dose-dependent, voltagedependent blockade of this voltage-gated sodium channel. f Dose-dependent, frequency-dependent blockade of this N-type calcium channel. g Dosedependent opening of this voltage-gated potassium channel.

spread and elevating seizure threshold in preclinical animal models, 4a has the potential to be more effective than topiramate and several other marketed antiepileptic drugs. The anticonvulsant profiles and favorable side effect properties of 4a suggest that this compound may be applicable to treating multiple forms of epilepsy (generalized tonic-clonic, complex partial, and absence seizures), including refractory (or pharmacoresistant) epilepsy, at dosing levels that confer a good margin of safety (therapeutic index of g20). Sulfamide 4a was nominated for clinical development. After numerous studies in preclinical development, including 2-week toxicology studies in rats and dogs at suprapharmacological oral doses, this drug candidate advanced into human clinical studies. Experimental Section General Chemical Procedures. Details for general methods are provided in our previous papers.6,8 Methodology for preparing various sulfamide products is also discussed in our previous papers.6,8 Synthetic examples are given for 4a and 4g by way of illustration. Flash-column chromatography was performed with silica gel. The structures of all new compounds were consistent with their 1H NMR and EI-MS mass spectra. Elemental microanalysis was performed by either Robertson Microlit Laboratories, Inc., Madison, NJ, or Quantitative Technologies, Inc. (QTI), Whitehouse, NJ. N-((Benzo[b]thien-3-yl)methyl)sulfamide (4a). Benzothiophene-3-carboxaldehyde (1.62 g, 10.0 mmol) was dissolved in anhydrous ethanol (50 mL). Sulfamide (4.0 g, 42 mmol) was added, and the mixture was heated to reflux for 16 h. After the mixture was cooled to room temperature, sodium borohydride (0.416 g, 11.0 mmol) was added and the mixture was stirred at room temperature for 3 h. The mixture was diluted with water (50 mL) and extracted with chloroform (375 mL). The extracts were concentrated and flash-column-chromatographed (5% methanol in CH2Cl2) to yield sulfamide 5 as a white solid (1.73 g, 72%). 1H NMR (DMSO-d6) δ 4.31 (2H, d, J = 6.3 Hz), 6.72 (2H, s), 7.08 (1H, t, J=6.3 Hz), 7.36-7.45 (2H, m), 7.62 (1H, s), 7.92 (1H, dd, J=6.6, 2.4 Hz), 7.98 (1H, dd, J=6.5, 2.3 Hz); MS (EI) m/z 241.06 [M - H]. Anal. Calcd for C9H10N2O2S2: C, 44.61; H, 4.16; N, 11.56; S, 26.47. Found: C, 44.54; H, 4.18; N, 11.60; S, 26.36. (5-Fluorobenzo[b]thien-3-yl)methylamine and N-[(5-Fluorobenzo[b]thien-3-yl)methyl]sulfamide (4g). 5-Fluoro-3-methylbenzothiophene (10.52 g, 63.3 mmol), benzoyl peroxide (1.53

g, 6.33 mmol), and N-bromosuccinimide (13.52 g, 75.9 mmol) were combined in chloroform (150 mL), and the mixture was heated to reflux for 3 h. The yellow solution was cooled and washed with water, and the organic solution was concentrated to a yellow solid. The solid was dissolved in anhydrous DMF (100 mL), and sodium azide (20.5 g, 315 mmol) was added. The mixture was heated to 50 C for 2 h. The mixture was cooled and then filtered. The solid was washed with ethyl acetate, and the combined organic solutions were concentrated to ∼50 mL and then diluted with water (100 mL) and diethyl ether (100 mL). The layers were separated, and the organic layer was then washed with brine (2  100 mL). The organic phase was concentrated to an orange oil and dissolved in a mixture of THF (250 mL) and water (25 mL). Triphenylphosphine (30.16 g, 115 mmol) was added, and the mixture was stirred at room temperature for 16 h. The solution was concentrated to a yellow solid, diluted with ethyl acetate (200 mL), and extracted with 1 N HCl (3  200 mL). The acidic extracts were neutralized with 6 N NaOH and then extracted with ethyl acetate (3  250 mL). The organic extracts were concentrated to an orange oil (8.7 g, 48 mmol, 76% yield) that was used in the next reaction without further purification. 1H NMR (CDCl3) δ 7.77 (1H, dd, J=8.6, 4.9 Hz), 7.4-7.7 (2H, m), 7.11 (1H, td, J=12.5, 2.5 Hz), 4.08 (2H, s). The (5-fluorobenzo[b]thien-3-yl)methylamine (8.7 g, 48 mmol) was dissolved in anhydrous 1,4-dioxane (200 mL), and sulfamide (20 g, 208 mmol) was added. The mixture was refluxed for 3 days, cooled to room temperature, and concentrated to a yellow solid. Water (250 mL) and dichloromethane (250 mL) were added. The solid that did not dissolve was collected by filtration. The filtrate was separated into layers, and the organic phase was washed with water (2  250 mL), concentrated to a solid, and combined with the solid collected by filtration. The combined solids were washed sequentially with water and cold dichloromethane to yield white solid 4g (8.87 g, 34.1 mmol, 71% yield). 1H NMR (CD3OD) δ 7.85 (dd, 1H, J= 6.6, 3.6 Hz), 7.66 (dd, 1H, J=7.4, 1.8 Hz), 7.62 (s, 1H), 7.15 (t, 1H, J=9.5 Hz), 4.21 (s, 2H). Anal. Calcd for C9H9FN2O2S2: C, 41.53; H, 3.48; N, 10.76; S, 24.64. Found: C, 41.55; H, 3.51; N, 10.73; S, 24.63. N-(Benzo[b]thien-3-yl)ethyl]sulfamide (5). 3-Acetylthianaphthene (3.00 g, 17.0 mmol) was added to a mixture of formic acid (10 mL) and formamide (10 mL), and the solution was heated to 150 C for 8 h. The mixture was cooled to room temperature, diluted with water (50 mL), and extracted with diethyl ether (3  50 mL). The ether extracts were washed with saturated aqueous NaHCO3 and brine and the solution was concentrated to afford crude product, which was flash-chromatographed

7534 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23

(5% methanol in CH2Cl2) to yield N-(1-benzo[b]thien-3ylethyl)formamide (1.76 g) as a white solid. This was suspended in concentrated HCl (30 mL), and the mixture was heated to reflux for 1.5 h, then diluted with water (100 mL) and then neutralized with 3 N NaOH until the pH was 14. The mixture was extracted with diethyl ether (3  100 mL), dried (magnesium sulfate), and concentrated to an orange oil. The oil was dissolved in anhydrous 1,4-dioxane (75 mL), and excess sulfamide was added. The mixture was heated to reflux for 2 h and then diluted with water (50 mL). The solution was extracted with ethyl acetate (2  50 mL), dried with magnesium sulfate, concentrated, and chromatographed (2.5-5% MeOH in CH2Cl2) to yield 5 as a white solid. 1H NMR (CD3OD) δ 8.01 (1H, dd, J=5.5, 0.7 Hz), 7.85 (1H, dt, J=6.0, 0.6 Hz), 7.49 (1H, s), 7.31-7.40 (2H, m), 4.95 (1H, q, J=5.1 Hz), 1.67 (3H, d, J=5.1 Hz). Anal. Calcd for C10H12N2O2S2: C, 46.85; H, 4.72; N, 10.93; S, 25.02. Found: C, 46.69; H, 4.81; N, 10.83; S, 24.83. Maximal Electroshock Seizure (MES) Test in Mice and Rats. The MES test procedure described by Swinyard et al. was employed.12 In experiments with mice, a 60-Hz current of 50-mA intensity was applied through corneal electrodes for a 0.2-s duration; with rats, seizure activity was induced by delivery of a 150-mA current (0.2 s) to the cornea. Both procedures caused immediate hindlimb tonic extension. Absence of tonic extension suggests that the test compound is able to prevent the spread of seizure discharge in neural tissue. Efficacy in this model is very predictive of clinical utility against tonic and/or clonic generalized seizures. Male CD-1 mice (Charles River Laboratories, 22-28 g) were fasted overnight prior to administering test compounds. Compounds were administered orally (po) as a suspension in 0.5% aqueous methylcellulose at various time points (0.5-8 h) prior to seizure testing. Ten mice per dose and 11 doses were used to establish ED50 values, i.e., the calculated dose required to block the hindlimb tonic-extensor component of the maximal electroshock seizure in 50% of the mice tested. The ED50 values, determined at the time of peak activity, were calculated by nonlinear regression using the Sigmoidal Emax model (Pharsight WinNonlin Program). Additional MES testing was performed as described above (by NeuroAdjuvants) except with CF-1 mice (Charles River Laboratories, 18-25 g), and the ED50 values were determined at 2 h following oral (po) administration. In studies conducted at the NINDS, male Sprague-Dawley rats (Simonsen Laboratories, 100-150 g) were deprived of food just prior to testing. Eight rats per dose and four doses (test compound in 0.5% aqueous methylcellulose) were used to establish ED50 values, which were determined at the time of peak activity after oral administration. Chemically Induced Seizures in Mice.12,17,25 These studies were conducted at the NINDS. Three convulsant compounds, bicuculline (Bic), picrotoxin (Pic), and pentylenetetrazol (PTZ), were used to induce seizures in male CF-1 albino mice (Charles River Laboratories, 18-25 g). Fifteen minutes postdosing with vehicle (0.5% aqueous methylcellulose) or test compound, Bic (2.7 mg/kg), Pic (3.15 mg/kg), or PTZ (85 mg/kg) was administered by subcutaneous (sc) injection at doses calculated to induce forelimb clonic seizures for 3 s in 97% of the mice (CD97). Animals not displaying a clonic seizure within the prescribed time frame, 30 min (Bic or PTZ) or 45 min (Pic), were considered protected. Eight mice per dose and a minimum of four doses were used to establish ED50 values. Anticonvulsant activity against these three convulsants indicates an ability to protect against threshold seizures, i.e., to raise the seizure threshold. The intravenous pentylenetetrazol (iv PTZ) seizure threshold test was also performed.26 In this case, the test compound does not have to completely prevent seizure occurrence but just delay its appearance, indicating the ability to modify seizure threshold. The iv PTZ test can determine if a compound can increase (anticonvulsant) or decrease (proconvulsant) seizure threshold.

Parker et al.

Two doses of the compound were employed, the first dose corresponding to the ED50 for protection against MES seizures and the second dose corresponding to the median toxic dose (TD50, dose that produces rotorod impairment in 50% of mice tested). Nine mice per dose were injected ip with vehicle (0.5% aqueous methylcellulose) or test compound. Fifteen minutes postdosing, a convulsant solution of PTZ (0.5% PTZ in 0.9% saline containing 10 USP units/mL of heparin sodium) was infused into the tail vein at a constant rate of 0.34 mL/min. The time in seconds from the start of the infusion to the appearance of the first twitch and the onset of clonus was recorded. The times to each end point were converted to mg/kg of PTZ for each mouse in the vehicle and test compound groups. An increase in the dose of PTZ to produce a first twitch or clonic seizure suggests that the test compound can increase seizure threshold (anticonvulsant). Audiogenic Seizures in Mice.27 These studies were conducted via the National Institute of Neurological Disorders and Stroke (NINDS) unit of the NIH. The ability of the test compound to prevent sound-induced seizures was evaluated in adult male and female Fring’s AGS mice (20-25 g; in-house breeding colony at the University of Utah, Salt Lake City). Individual mice were placed into a Plexiglas cylinder (diameter, 15 cm; height, 18 cm) fitted with an audiotransducer and exposed to a sound stimulus of 110 dB (11 kHz) delivered for 20 s. Sound-induced seizures are characterized by wild running followed by loss of righting reflex with forelimb and hindlimb tonic extension. Drug-treated mice that did not display hindlimb tonic extension were considered protected. There were eight mice per dose and a minimum of four doses, which established an ED50 value at the time to peak effect. Test compound or vehicle (0.5% aqueous methylcellulose) was administered ip 1 h prior to sound induction. Kindling Studies in Rats.17 Adult male Sprague-Dawley rats (300-400 g) were surgically implanted with bipolar electrodes placed in the hippocampus. Rats were kindled by repetitive electrical stimulation (50 Hz, 10-s train of 1 ms, biphasic 200-μA pulses every 30 min for 6 h every other day for a total of 60 stimulations) resulting in stage 5 bilateral motor seizures. One week later, the rats received two to three suprathreshold stimulations every 30 min before treatment with test compound to ensure stability of the behavioral seizure stage and after-discharge duration. Fifteen minutes after the last stimulation, a single dose of vehicle or test compound was administered ip; after 15 min more, each rat was stimulated every 30 min for 34 h. After each stimulation, individual seizure scores and afterdischarge durations were recorded. The group scores (mean ( SEM) were calculated for each parameter, with eight rats per dose and at least four doses being used to establish the ED50 values. Efficacy was measured in terms of the ability of a compound to modify the seizure score (severity of spread) and afterdischarge duration (excitability) of the generalized seizures. Voltage-Gated Naþ Channels.28 Sodium channel activity was studied by using a whole-cell patch-clamp technique in CHL1610 cells that stably express rat Nav1.2. A 45-s preconditioning pulse (at -107 and -67 mV) was followed by 3 s of brief (5-ms) depolarizations to -7 mV at 10 Hz. The membrane potential during the time between each depolarization was the same as the preconditioning voltage. The interval between each preconditioning pulse was 15 s, during which time the cell was held at -107 mV. The extracellular solution perfusing the cell contained NaCl (132 mM), KCl (5.4 mM), CaCl2 (1.8 mM), MgCl2 (0.8 mM), HEPES (10 mM), and glucose (10 mM) at pH 7.4. The pipet solution contained CsCl (45 mM), CsF (100 mM), EGTA (5.0 mM), HEPES (10 mM), and glucose (5.0 mM) at pH 7.4. Data were acquired in the absence and presence (after 2-3 min application) of the test compound. All experiments were performed at 22 C. The peak current amplitude during the 30th 5-ms depolarization to -7 mV was used to determine percent inhibition by the compounds tested. To obtain the 50% inhibitory concentration (IC50) value, the concentration-response

Article

data were fitted to a logistic function of the form R=100 - 100/ (1 þ C/IC50)p, where R is the percentage inhibition, p is the slope coefficient, and C is the concentration of the test compound. Voltage-Gated N-Type Ca2þ Channels.28c,29 Compounds were prepared as 1 M solutions in DMSO and diluted to the indicated concentrations in either “patch-clamp control buffer,” containing 121 mM triethylamine-HCl, 10 mM BaCl2, 1.0 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4), or for calcium-imaging studies, Hank’s balanced salt solution (Invitrogen), to which was added 2.5 mM CaCl2, 20 mM HEPES, and 0.1% bovine serum albumin. MVIIA was prepared as a 200 μM stock solution in patch-clamp control buffer containing 0.3% bovine serium albumin. The N-type voltage-gated calcium channel stable cell line was generated with HEK cells by expressing rat Cav2.2 (R1B) subunit in pcDNA3.1 (Genbank no. AAO53230) and R2δ and β3 in pBudCE4.1 vectors under selection by 400 μg/mL G418 and 200 μg/mL Zeocin, respectively. Cells were clonally isolated, expanded, and screened by Western blot analyses and then further tested for expression of characteristic N-type calcium currents by whole-cell patchclamp. Rat Cav2.2-containing channels were tested by using a calcium indicator dye detection system (FDSS, Hamamatsu) and the whole-cell patch-clamp technique (EPC-10 amplifier with Pulse software, HEKA). For the FDSS assay, peak responses to 50 mM KCl stimulation were recorded. Responses in the presence of different concentrations of test compounds were normalized to the peak control response to 50 mM KCl (set to 100%) and the peak response in the presence of 200 nM MVIIA (set to 0%). The data from four wells for each concentration were averaged, and the averaged points were subjected to a nonlinear regression curve fit (GraphPad Prism) to determine the IC50 values. For whole-cell patch-clamp, current (in pA) was measured in a 20-ms window surrounding the peak current evoked during the step to þ20 mV for each pulse. The amount of current at the end of a given drug concentration was normalized to the control level determined at the start of the experiment. The percent inhibition of a given concentration of test compound was calculated as 100 - [100  (pA in compound)/(pA in control)]. For determining the IC50, the percent inhibition at each concentration (1, 10, 30, 100, and 300 μM in 3, 6, 10, 8, and 5 cells, respectively) was averaged across all cells tested at that concentration, and the average data for all concentrations tested were fit to the same logistic function as above. Where applicable, data were analyzed by a Student’s t test and are expressed as the mean ( SEM. Neurotoxicity in Mice.30 To determine sedative and/or ataxic side effects for the test compounds, the standard rotorod toxicity test was performed. The time of peak effect for minimal motor impairment on the rotorod was determined following administration of 100 mg/kg (ip) of compound. Mice were observed by a trained technician on a rotating rotorod (6 rpm) for 1 min to check for motor impairment, ataxia, or other signs of behavioral toxicity. Motor impairment was defined as the inability of a mouse to maintain equilibrium for 60 s in three consecutive trials on the rotorod. Carbonic Anhydrase Inhibition. The inhibition of CA-II was performed by the pH-shift method, which involves the hydration of CO2. We have described the procedure for this assay in earlier papers.8a-8d An analogous assay method was performed by Cerep, an independent contract laboratory.13,14

Acknowledgment. We are grateful to the National Institute of Neurological Disorders and Stroke (NINDS) for conducting numerous studies involving anticonvulsant pharmacological models, with special thanks given to James Stables. We thank Geert Mannens, Anthony Streeter, Heng-Keang Lim, and Jose Silva for contributions to the drug metabolism studies, Lisa Minor for some carbonic anhydrase-II measurements, and Tasha Hutchinson for helping with the cell culture work.

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Supporting Information Available: Table of microanalytical data for new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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