Ex Parte Ryde et alDownload PDFBoard of Patent Appeals and InterferencesMar 10, 201110895405 (B.P.A.I. Mar. 10, 2011) Copy Citation UNITED STATES PATENT AND TRADEMARK OFFICE UNITED STATES DEPARTMENT OF COMMERCE United States Patent and Trademark Office Address: COMMISSIONER FOR PATENTS P.O. Box 1450 Alexandria, Virginia 22313-1450 www.uspto.gov APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO. 10/895,405 07/21/2004 Tuula A. Ryde 029318-1027 6737 31049 7590 03/11/2011 Elan Drug Delivery, Inc. c/o Foley & Lardner 3000 K Street, N.W. Suite 500 Washington, DC 20007-5109 EXAMINER ALSTRUM ACEVEDO, JAMES HENRY ART UNIT PAPER NUMBER 1616 MAIL DATE DELIVERY MODE 03/11/2011 PAPER Please find below and/or attached an Office communication concerning this application or proceeding. The time period for reply, if any, is set in the attached communication. PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE __________ BEFORE THE BOARD OF PATENT APPEALS AND INTERFERENCES __________ Ex parte TUULA A. RYDE, DOUGLAS C. HOVEY, and H. WILLIAM BOSCH __________ Appeal 2010-011978 Application 10/895,405 Technology Center 1600 __________ Before LORA M. GREEN, MELANIE L. MCCOLLUM, and JEFFREY N. FREDMAN, Administrative Patent Judges. FREDMAN, Administrative Patent Judge. DECISION ON APPEAL1 This is an appeal under 35 U.S.C. § 134 involving claims to a nanoparticulate sildenafil free base composition. We have jurisdiction under 35 U.S.C. § 6(b). We affirm-in-part and enter a new ground of rejection. 1 The two-month time period for filing an appeal or commencing a civil action, as recited in 37 C.F.R. § 1.304, or for filing a request for rehearing, as recited in 37 C.F.R. § 41.52, begins to run from the “MAIL DATE” (paper delivery mode) or the “NOTIFICATION DATE” (electronic delivery mode) shown on the PTOL-90A cover letter attached to this decision. Appeal 2010-011978 Application 10/895,405 2 Statement of the Case Background “The present invention relates to compositions of sildenafil free base having an effective average particle size of less than about 2 microns. Preferably, the compositions also comprise at least one surface stabilizer associated with the surface of the sildenafil free base particles” (Spec. 1 ¶ 0002). The Claims Claims 1, 2, 4, and 6-39 are on appeal.2 Claims 1 and 27 are representative, and the remaining claims have not been argued separately and therefore stand or fall together with claims 1 and 27. 37 C.F.R. § 41.37(c)(1)(vii). Claims 1 and 27 read as follows: 1. A nanoparticulate sildenafil free base composition comprising: (a) particles of sildenafil free base having an effective average particle size of less than 2000 nm; and (b) at least one surface stabilizer, wherein the composition is bioequivalent when administered under fed and fasted conditions. 27. The composition of claim 24, wherein the non-sildenafil free base PDE 5 inhibitor is selected from the group consisting of vardenafil, tadalafil, TA-1790, UK-114542, Compound 14, EMD221829, EMR 62 203, T-1032, M-54033, M- 54018, and E- 4010. 2 Claims 40-45, 47, 49-69, 71, 73-101, and 103-107 were withdrawn from consideration as drawn to a non-elected invention in the Examiner Action mailed Nov. 23, 2009. Appeal 2010-011978 Application 10/895,405 3 The issues A. The Examiner rejected claims 27-29 under 35 U.S.C. § 112, first paragraph as failing to comply with the written description requirement (Ans. 4-5). B. The Examiner rejected claims 27-29 under 35 U.S.C. § 112, first paragraph, as not enabled for the compositions disclosed in claim 27 (Ans. 6-8). C. The Examiner rejected claims 1, 2, 4, 6-21, 24, 26, and 30-39 under 35 U.S.C. § 103(a) as obvious over Straub3 and Bell-Huff4 (Ans. 9-13). D. The Examiner rejected claims 22, 23, 25, 28, and 29 under 35 U.S.C. § 103(a) as obvious over Straub, Bell-Huff, and Maggi5 (Ans. 13-15). E. The Examiner rejected claims 1, 2, 4, 6-21, and 33-39 under 35 U.S.C. § 103(a) as obvious over Liversidge,6 Bell-Huff, and DIH7 (Ans. 15- 18). F. The Examiner provisionally rejected claims 1, 2, 4, 6-24, and 30-39 on the ground of nonstatutory obviousness-type double patenting over claims 1, 10, 11, 15-26, 31, and 41 of copending US Application 10/619,539 and Merck8 (Ans. 18-19). 3 Straub et al., US 2002/0142050 A1, published Oct. 3, 2002. 4 Bell-Huff et al., US 2002/0002172 A1, published Jan. 3, 2002. 5 Maggi et al., Erectile dysfunction: from biochemical pharmacology to advances in medical therapy, 143 EUROPEAN JOURNAL OF ENDOCRINOLOGY 143-54 (2000). 6 Liversidge et al., US 6,267,989 B1, issued Jul. 31, 2001. 7 Lacy et al., 1999 DRUG INFORMATION HANDBOOK 926-927, Lexi- Comp, Inc., Hudson, Ohio (1999) 8 Merck Index, 13th Edition, Entry 8563, 1523 (2001). Appeal 2010-011978 Application 10/895,405 4 G. The Examiner provisionally rejected claims 1, 2, 4, 6-22, 24, and 30- 39 on the ground of nonstatutory obviousness-type double patenting over claims 1-24 of copending US Application 10/701,064 and Merck (Ans. 20- 21). A. U.S.C. § 112, first paragraph, written description The Examiner finds that the ordinary skilled artisan would reasonably conclude that Appellants were not in possession of TA-1790, UK-114542, Compound 14, EMD221829, EMR 62 203, T-1032, E- 4010 M-54033, and M-54018 are not known in the prior art and Appellants' do not identify said compounds with any chemical structures, chemical abstract registry numbers, IUPAC chemical names, etc. and Appellants do not disclose how to obtain said compounds (Ans. 5). Appellants contend that “[e]ach of TA-1790, UK-114542, Compound 14, EMD221829, EMR 62203, T-I032, E-4010[,] M-54033, and M-54018 were known in the art as PDE 5 inhibitors prior to the July 23, 2003, priority date of the present application. Evidence in this regard was submitted in the response filed November 9, 2009, as Exhibits 1-5” (App. Br. 9). Appellants contend that they “are merely showing that the recited compounds would be recognized as PDE 5 inhibitors by the person of ordinary skill in the art, at the time of filing. This is consistent with Capon, id. and is analogous to a trademark or tradename being recognized as descriptive of a specific composition” (App. Br. 10). Appeal 2010-011978 Application 10/895,405 5 The issue with respect to this rejection is: Does the evidence of record support the Examiner’s conclusion that Appellants lacked possession of the compounds recited in claim 27? Findings of Fact 1. Exhibits 1-5 teach descriptions of PDE5 inhibitors with the particular claimed names (see Exhibits 1-5). Principles of Law Falko-Gunter teaches that “we hold that where, as in this case, accessible literature sources clearly provided, as of the relevant date, genes and their nucleotide sequences (here ‘essential genes’), satisfaction of the written description requirement does not require either the recitation or incorporation by reference (where permitted) of such genes and sequences.” Falko-Gunter Falkner v. Inglis, 448 F.3d 1357, 1368 (Fed. Cir. 2006). Analysis Consistent with Falko-Gunter, Appellants have demonstrated that accessible literature sources provide descriptions of the compounds being claimed (FF 1). We note that claim 27 requires that the named compounds are PDE5 inhibitors, which limits the universe of compounds. We appreciate the Examiner’s concern that mention “in a single reference does not establish that th[ese] compound[s] were well known” (Ans. 23), but the court did not establish a requirement that descriptive support of specifically named compounds requires mention in more than one literature source or incorporation by reference, where the compounds were known in the prior art. See Falko-Gunter Falkner, 448 F.3d at 1368. Appeal 2010-011978 Application 10/895,405 6 Conclusion of Law The evidence of record does not support the Examiner’s conclusion that Appellants lacked possession of the compounds recited in claim 27. B. U.S.C. § 112, first paragraph, enablement The Examiner finds that: It would be impossible for the ordinary skilled artisan to prepare compositions comprising TA-1790, UK- 114542, Compound 14, EMD221829, EMR 62 203, M-54033, and M-54018, because TA-1790, UK-114542, Compound 14, EMD221829, EMR 62 203, T-1032, E-4010 M-54033, and M-54018, because the ordinary skilled artisan based upon the teachings of the art and Appellants’ disclosure would not know what these compounds are. (Ans. 8). Appellants contend that “the compounds recited in claim 27 are all known as PDE 5 inhibitors. Moreover, because these compounds share a common activity, inhibiting PDE 5, one that is shared with the clinically approved PDE5 inhibitors sildenafil, tardanafil and vardanafil, the use of such compounds by one of ordinary skill in the art is enabled” (App. Br. 12). The issue with respect to this rejection is: Does the evidence of record support the Examiner’s conclusion that use of the compounds recited in claim 27 would have required undue experimentation? Principles of Law When rejecting a claim under the enablement requirement of section 112, the PTO bears an initial burden of setting forth a reasonable explanation as to why it believes that the scope of protection provided by that claim is not adequately enabled by the description of the invention provided in the specification of the application. Appeal 2010-011978 Application 10/895,405 7 In re Wright, 999 F.2d 1557, 1561-62 (Fed. Cir. 1993). “[T]he question of undue experimentation is a matter of degree. The fact that some experimentation is necessary does not preclude enablement; what is required is that the amount of experimentation ‘must not be unduly extensive.”’ PPG Indus., Inc. v. Guardian Indus. Corp., 75 F.3d 1558, 1564 (Fed. Cir. 1996). Findings of Fact 2. Brandle9 teaches “[i]nvestigations of trans-4-[4-(3-chloro-4- methoxybenzylamino)benzo[4,5]-thieno[ 2,3-d]pyrimidin-2- yl]cyclohexanecarboxylic acid, ethanolamine salt (EMD 221829) for haemodynamics and for erectile function in various animal species, in some cases with simultaneous administration of nitrate, compared with sildenafil citrate” (Brandle 4 ¶ 0103). Analysis The Examiner’s argument is premised on the inability of the ordinary artisan to “know what these compounds are” (Ans. 8). However, in view of Appellants’ Evidence Appendix, this argument is not tenable. For example, compound EMD 221829 is disclosed in Brandle immediately adjacent to its chemical name (FF 2). The Examiner has provided no evidence that undue experimentation would have been required to perform the literature search for these PDE5 inhibitors and to then know the chemical composition of these compounds. 9 Brandle et al., US 2003/0022906 A1, published Jan. 30, 2003. Appeal 2010-011978 Application 10/895,405 8 Conclusion of Law The evidence of record does not support the Examiner’s conclusion that use of the compounds recited in claim 27 would have required undue experimentation. C. 35 U.S.C. § 103(a) over Straub and Bell-Huff The Examiner finds that “Straub teaches porous drug matrices and method of manufacture thereof, wherein the drugs are especially low aqueous solubility drugs that are combined with hydrophilic or hydrophobic excipients in said matrix, wherein the excipients inhibit or prevent crystallization” (Ans. 10). The Examiner finds that “[s]uitable drugs for incorporation into the drug matrices are drugs with a solubility of less than about 10 mg/ml in water [0027] and include sildenafil citrate as a preferred drug” (Ans. 10). The Examiner finds that Straub teaches that the “matrix must contain drug particles that have a particle size between about 100 nm and 5 microns, preferably between about 500 nm and 5 microns” (Ans. 10). The Examiner finds that “Bell-Huff teaches pharmaceutical formulations of sildenafil in the form of its free base” (Ans. 11). The Examiner finds it obvious to “utilize sildenafil free base, because sildenafil free base does not result in formulations with an undesirable taste compared to sildenafil citrate formulations” (Ans. 11). Appellants contend that the “mere recitation of 111 species in Straub is insufficient, therefore, to provide, even sildenafil citrate. This is not the only failing of Straub, however. Even if the person of ordinary [skill] found a reason to select sildenafil citrate from the broad list of compounds in Appeal 2010-011978 Application 10/895,405 9 Straub, there is no evidence that Straub enables a method of making nanoparticulate sildenafil citrate or sildenafil free base” (App. Br. 18). Appellants contend that “there is no reason to form it into the nanoparticles disclosed by Straub for the purpose of improving dissolution and bioavailability. Why would a person of ordinary skill in the art have a reason to improve the dissolution and bioavailability of a drug (Straub) when the art teaches that the drug is already efficiently absorbed (Bell-Huff)?” (App. Br. 19). The issue with respect to this rejection is: (i) Does the evidence of record support the Examiner’s conclusion that Straub and Bell-Huff suggest a “nanoparticulate sildenafil free base composition” as required by Claim 1? (ii) And if yes, have Appellants presented evidence of secondary considerations, that when weighed with the evidence of obviousness, are sufficient to support a conclusion of non-obviousness? Findings of Fact 3. Straub teaches formulations of drugs, especially drugs having low solubility, and more particularly to methods of making formulations of such drugs to enhance their rate of dissolution, and optionally, to enhance their stability through the inclusion of hydrophobic or hydrophilic excipients that enhance dissolution rate, stabilize drug in crystalline form by inhibiting crystal growth or stabilize drug in amorphous form by preventing crystallization. (Straub 1 ¶ 0002). Appeal 2010-011978 Application 10/895,405 10 4. Straub teaches that “[p]referred drugs include . . . sildenafil citrate” (Straub 4-5 ¶ 0072). 5. Straub teaches that the “matrices may contain hydrophilic or hydrophobic excipients such as polymers, including water soluble polymers, amino acids or sugars which can serve as bulking agents or as wetting agents, wetting agents such as surfactants” (Straub 5 ¶ 0074). Straub teaches that polymers can include gelatin (Straub 5 ¶ 0078). 6. Straub teaches the “form of the drug matrix (dry powder) is critical to the dissolution rate. The matrix must contain microparticles of drug, which preferably have a diameter between about 100 mn [sic nm] and 5 μm, more preferably between about 500 nm and 5 μm” (Straub 2 ¶ 0024). 7. Bell-Huff teaches “pharmaceutical formulations of the compound sildenafil and in particular to rapidly disintegrating oral dosage forms which contain sildenafil in the form of its free base” (Bell-Huff 1 ¶ 0002). 8. Bell-Huff teaches that: One problem with the rapidly dispersing solid dosage forms described above is that the patient will taste the pharmaceutically active substance as the dosage form disintegrates in the mouth. For some pharmaceutically active substances, the taste, if only slightly unpleasant, can be rendered acceptable by the use of sweetening agents or flavoring agents to mask the taste. However, this technique does not completely mask the taste of sildenafil citrate. (Bell-Huff 1 ¶ 0006). 9. Bell-Huff teaches that “we have surprisingly discovered that sildenafil, in the form of its free base has extremely low solubility in water, Appeal 2010-011978 Application 10/895,405 11 and in saliva, and this makes it particularly suitable for use in orally dispersible formulations, being virtually tasteless” (Bell-Huff 1 ¶ 0007). 10. The Specification teaches that Viagra® administered under fed and fasted conditions exhibited an AUCinf of 2514 ng/mL·h and 1749.6 ng/mL·h, respectively - a difference of 764.4 ng/mL·h or ~ 30%. The nanoparticulate sildenafil citrate composition administered under fed and fasted conditions exhibited an AUCinf of 1335.2 ng/mL·h and 897.8 ng/mL·h, respectively - a difference of 437.4 ng/mL·h or ~ 33%. In surprising contrast to these results, the nanoparticulate sildenafil free base composition, administered under fed and fasted conditions, exhibited an AUCinf of 2127.8 ng/mL·h and 2105.3 ng/mL·h, respectively - a difference of 22.5 ng/mL·h ~ or 1%. (Spec. 65 ¶ 0200). Principles of Law “The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007). “[E]ven though applicant’s modification results in great improvement and utility over the prior art, it may still not be patentable if the modification was within the capabilities of one skilled in the art, unless the claimed ranges ‘produce a new and unexpected result which is different in kind and not merely in degree from the results of the prior art.” In re Huang, 100 F.3d 135, 139 (Fed. Cir. 1996). Analysis Straub teaches improved drug formulations (FF 3), and in particular, that the “form of the drug matrix (dry powder) is critical to the dissolution Appeal 2010-011978 Application 10/895,405 12 rate. The matrix must contain microparticles of drug, which preferably have a diameter between about 100 mn and 5 μm, more preferably between about 500 nm and 5 μm” (Straub 2 ¶ 0024; FF 6). Straub teaches that the formulations may include stabilizers such as surfactants or gelatin (FF 5). Straub teaches that “[p]referred drugs include . . . sildenafil citrate” (Straub 4-5 ¶ 0072; FF 4). Bell-Huff teaches “pharmaceutical formulations of the compound sildenafil and in particular to rapidly disintegrating oral dosage forms which contain sildenafil in the form of its free base” (Bell-Huff 1 ¶ 0002; FF 7). Bell-Huff teaches that: One problem with the rapidly dispersing solid dosage forms described above is that the patient will taste the pharmaceutically active substance as the dosage form disintegrates in the mouth. For some pharmaceutically active substances, the taste, if only slightly unpleasant, can be rendered acceptable by the use of sweetening agents or flavoring agents to mask the taste. However, this technique does not completely mask the taste of sildenafil citrate. (Bell-Huff 1 ¶ 0006; FF 8). Bell-Huff teaches that “we have surprisingly discovered that sildenafil, in the form of its free base has extremely low solubility in water, and in saliva, and this makes it particularly suitable for use in orally dispersible formulations, being virtually tasteless” (Bell-Huff 1 ¶ 0007; FF 9). Applying the KSR standard of obviousness to the findings of fact, we agree with the Examiner that the person of ordinary creativity would have had specific reasons to substitute the sildenafil free base form taught by Bell-Huff for the sildenafil citrate form used in Straub, specifically to Appeal 2010-011978 Application 10/895,405 13 minimize the unpleasant taste of the drug (FF 8-9). Such a combination is merely a “predictable use of prior art elements according to their established functions.” KSR, 550 U.S. at 417. Appellants contend that the “mere recitation of 111 species in Straub is insufficient, therefore, to provide even sildenafil citrate. This is not the only failing of Straub, however. Even if the person of ordinary [skill] found a reason to select sildenafil citrate from the broad list of compounds in Straub, there is no evidence that Straub enables a method of making nanoparticulate sildenafil citrate or sildenafil free base” (App. Br. 18). We are not persuaded. Straub expressly teaches the use of sildenafil citrate (FF 4). That the prior art “discloses a multitude of effective combinations does not render any particular formulation less obvious. This is especially true because the claimed composition is used for the identical purpose taught by the prior art.” Merck & Co. v. Biocraft Labs., Inc., 874 F.2d 804, 807 (Fed. Cir. 1989). In Gleave, the Federal Circuit expressly addressed the issue of lists of large numbers of compounds, where the “list includes more than 1400 sequences.” In re Gleave, 560 F.3d 1331, 1333 (Fed. Cir. 2009). The Court, found that “Wraight expressly lists every possible fifteen-base-long oligodeoxynucleotide sequence in IGFBP-2, and under our precedent, this list anticipates Gleave’s claims.” Id. at 1338. Consequently, we find that the combination of Straub and Bell-Huff reasonably renders claim 1 obvious. Appellants contend that “there is no reason to form it into the nanoparticles disclosed by Straub for the purpose of improving dissolution and bioavailability. Why would a person of ordinary skill in the art have a Appeal 2010-011978 Application 10/895,405 14 reason to improve the dissolution and bioavailability of a drug (Straub) when the art teaches that the drug is already efficiently absorbed (Bell-Huff)?” (App. Br. 19). Appellants also contend that the nanoparticulate sildenafil free base “would be expected to be more soluble in saliva, and therefore less palatable” (App. Br. 19). We are not persuaded. The issue is whether it would have been obvious to substitute the free base form of sildenafil for sildenafil citrate in Straub. The Examiner reasonably finds it obvious to “utilize sildenafil free base, because sildenafil free base does not result in formulations with an undesirable taste compared to sildenafil citrate formulations” (Ans. 11; FF 8- 9). Appellants provide no evidence to rebut Bell-Huff’s teaching that the free base is more palatable. See In re Pearson, 494 F.2d 1399, 1405 (CCPA 1974) (“Attorney’s argument in a brief cannot take the place of evidence.”). Appellants contend that the “mere assertion that other drugs can be made nanoparticulate does not provide sufficient teachings as to how to make nanoparticulate sildenafil free base in view of the known unpredictability in the field of nanoparticulate drugs” (Reply Br. 4). We are not persuaded. Kubin stated that “[r]esponding to concerns about uncertainty in the prior art influencing the purported success of the claimed combination, this court [in O’Farrell] stated: ‘[o]bviousness does not require absolute predictability of success … all that is required is a reasonable expectation of success.”’ In re Kubin, 561 F.3d 1351, 1360 (Fed. Cir. 2009) (citing In re O’Farrell, 853 F.2d 894, 903-904 (Fed. Cir. 1988)). Here, there is a very reasonable expectation that given Straub’s specific teachings on making drugs nanoparticulate (see Straub 7-8 ¶¶ 0115- Appeal 2010-011978 Application 10/895,405 15 0119) and specific teaching of sildenafil citrate (FF 4), the ordinary artisan would reasonably be expected to be enabled to make nanoparticulate sildenafil. Straub exemplifies successful synthesis of 5 different drugs (Straub 9-10). Appellants have provided no evidence to suggest any real difficulties in performing this method. Appellants’ contention of the “known unpredictability” simply represents attorney argument. See In re Pearson, 494 F.2d 1399, 1405 (CCPA 1974) (“Attorney’s argument in a brief cannot take the place of evidence.”). Appellants contend that the finding that “a nanoparticulate sildenafil free base composition is bioequivalent when administered under fed and fasted conditions is not found anywhere in the art, nor is it a predictable result of practicing the art” (App. Br. 24). Appellants contend that “the variation between fed and fasted conditions is only 1% for sildenafil free base, but 33% for sildenafil citrate. See page 18, paragraph [0055]. This is completely unexpected. For the patient, the advantage of greatly reduced variability overcomes the disadvantage of using the less bioavailable sildenafil free base” (App. Br. 25). We are not persuaded. We agree with the Examiner that the result is not commensurate in scope with claim 1, which, in light of the limitation in claim 2 that an AUC difference between fed and fasted states of “less than 25%” are bioequivalent, reasonably encompasses the slightly broader situation where the difference of 33% is also treated as “bioequivalent”. More significantly, we also conclude that even if we treated the results in Table 4 as somewhat “unexpected”, the instant showing is insufficient to overcome the strong showing of obviousness in this case, where Straub and Appeal 2010-011978 Application 10/895,405 16 Bell-Huff provide reasoned guidance suggesting the claimed composition (FF 3-9). See Pfizer, Inc. v. Apotex, Inc., 480 F.3d 1348, 1372 (Fed. Cir. 2007) (“[W]e hold that even if Pfizer showed that amlodipine besylate exhibits unexpectedly superior results, this secondary consideration does not overcome the strong showing of obviousness in this case. Although secondary considerations must be taken into account, they do not necessarily control the obviousness conclusion. Newell Cos., Inc. v. Kenney Mfg. Co., 864 F.2d 757, 768 (Fed.Cir.1988)”). Conclusion of Law (i) The evidence of record supports the Examiner’s conclusion that Straub and Bell-Huff suggest a “nanoparticulate sildenafil free base composition” as required by Claim 1. (ii) Appellants have not presented evidence of secondary considerations, that when weighed with the evidence of obviousness, are sufficient to support a conclusion of non-obviousness. D. 35 U.S.C. § 103(a) over Straub, Bell-Huff, and Maggi The Examiner finds it obvious “to combine the teachings of Straub and Maggi, because both sildenafil and yohimbine are art-recognized pharmaceuticals indicated for the treatment of erectile dysfunction” (Ans. 14). The Examiner provides sound fact-based reasoning for combining the Maggi with Straub and Bell-Huff (see Ans. 14). We adopt the fact finding and analysis of the Examiner as our own. Appellants argue the underlying obviousness rejection over Straub and Bell-Huff, but Appellants do not identify any material defect in the Examiner's reasoning for combining Appeal 2010-011978 Application 10/895,405 17 Maggi with Straub and Bell-Huff. Since Appellants only argue the underlying rejection of Straub and Bell-Huff which we affirmed above, we affirm this rejection for the reasons stated by the Examiner. E. 35 U.S.C. § 103(a) over Liversidge, Bell-Huff, and DIH The Examiner finds that “Liversidge teaches nanoparticulate compositions and methods of making said compositions, wherein said compositions comprise a poorly soluble crystalline or amorphous drug and one or more non-crosslinked surface stabilizers adsorbed to the surface of the drug having an effective average particle size of from about 150 nm to about 350 nm” (Ans. 15). The Examiner finds that Liversidge teaches that “[s]uitable surface stabilizers include nonionic and ionic surfactants” (Ans. 15). The Examiner finds that Liversidge teaches that “suitable drugs include . . . vasodilators” (Ans. 15). The Examiner finds that the “teachings of Bell-Huff are set forth above. The DIH teaches that sildenafil is a vasodilator, because it induces smooth muscle relaxation in the corpus cavernosum and an inflow of blood” (Ans. 16). The Examiner finds it obvious “to modify the teachings of Liversidge with the teachings of Bell-Huff, because sildenafil free base is a low- solubility drug that is a known vasodilator (DIH); Liversidge's formulations are designed to utilize poor solubility drugs; and Liversidge explicitly identifies vasodilators as suitable drugs for use in the invented formulations” (Ans. 17). Appellants contend that the “broad genus of ‘vasodilators’ in Liversidge is insufficient to provide the specific elements found in the claim” Appeal 2010-011978 Application 10/895,405 18 (App. Br. 21). Appellants contend that the “references also teach away from a nanoparticulate formulation of sildenafil free base. Bell-Huff teaches away because nanoparticulate sildenafil free base would be more soluble than the non-nanoparticulate form and therefore less palatable, given that the lower solubility of the free base over the citrate underlies its greater palatability” (App. Br. 23). The issue with respect to this rejection is: (i) Does the evidence of record support the Examiner’s conclusion that Liversidge, DIH, and Bell-Huff suggest a “nanoparticulate sildenafil free base composition” as required by Claim 1? (ii) And if yes, have Appellants presented evidence of secondary considerations, that when weighed with the evidence of obviousness, are sufficient to support a conclusion of non-obviousness? Findings of Fact 11. Liversidge teaches nanoparticulate compositions comprising a poorly soluble crystalline or amorphous drug and one or more non- crosslinked surface stabilizers adsorbed to the surface of the drug, having an optimal effective average particle size of from about 150 nm to about 350 nm, more preferably from about 150 nm to about 300 nm, even more preferably from about 150 nm to about 250 nm, and most preferably from about 150 to about 200 nm. The compositions exhibit minimal particle aggregation and crystal growth following prolonged storage periods and/or exposure to elevated temperatures. (Liversidge, col. 3, ll. 41-51). Appeal 2010-011978 Application 10/895,405 19 12. Liversidge teaches that the “drug can be selected from a variety of known classes of drugs, including, for example . . .vasodilators” (Liversidge, col. 7, ll. 10-31). 13. Liversidge teaches that “surface stabilizers include various polymers, low molecular weight oligomers, natural products, and surfactants. Preferred surface stabilizers include nonionic and ionic surfactants. Two or more surface auxiliary stabilizers can be used in combination” (Liversidge, col. 7, ll. 39-42). 14. Liversidge teaches that “[n]anoparticulate compositions are superior to macro-sized particulate drug formulations, as nanoparticulate drug formulations can exhibit reduced toxicity and enhanced efficacy” (Liversidge, col. 1, ll. 19-23). 15. The Examiner finds that “DIH demonstrates that sildenafil is a known vasodilator” (Ans. 16). Analysis Liversidge teaches forming nanoparticulates of a variety of drugs, including vasodilators (FF 11-13). DIH teaches that sildenafil is a vasodilator (FF 14). Bell-Huff teaches that sildenafil free base is a superior form of the drug since it has improved palatability (FF 7-9). Applying the KSR standard of obviousness to the findings of fact, we agree with the Examiner that the person of ordinary creativity would have reasonably formulated the sildenafil free base form taught by Bell-Huff using the nanoparticulate form of Liversidge to minimize the unpleasant taste of the drug (FF 8-9) and since Liversidge teaches that “[n]anoparticulate compositions are superior to macro-sized particulate drug Appeal 2010-011978 Application 10/895,405 20 formulations, as nanoparticulate drug formulations can exhibit reduced toxicity and enhanced efficacy” (Liversidge, col. 1, ll. 19-23; FF 14). Such a combination is merely a “predictable use of prior art elements according to their established functions.” KSR, 550 U.S. at 417. Appellants contend that the “broad genus of ‘vasodilators’ in Liversidge is insufficient to provide the specific elements found in the claim” (App. Br. 21). We are not persuaded. While Liversidge broadly teaches the use of any vasodilator, DIH and Bell-Huff teach specific reasons to use sildenafil free base (FF 7-9). Further, Liversidge provides reasons why the nanoparticulate form is superior (FF 14). Thus, an ordinary artisan would have had reason to select the specific claimed elements of nanoparticulate and sildenafil free base. That the prior art “discloses a multitude of effective combinations does not render any particular formulation less obvious. This is especially true because the claimed composition is used for the identical purpose taught by the prior art.” Merck & Co. v. Biocraft Labs., Inc., 874 F.2d 804, 807 (Fed. Cir. 1989). Appellants contend that the “references also teach away from a nanoparticulate formulation of sildenafil free base. Bell-Huff teaches away because nanoparticulate sildenafil free base would be more soluble than the non-nanoparticulate form and therefore less palatable, given that the lower solubility of the free base over the citrate underlies its greater palatability” (App. Br. 23). We are not persuaded. Appellants have not identified any specific teaching in Bell-Huff which teaches away from nanoparticulate form. Like Appeal 2010-011978 Application 10/895,405 21 our appellate reviewing court, “[w]e will not read into a reference a teaching away from a process where no such language exists.” DyStar Textilfarben GmbH & Co. Deutschland KG v. C.H. Patrick Co., 464 F.3d 1356, 1364 (Fed. Cir. 2006). Appellants have also provided no evidence to rebut Bell- Huff’s teaching that the free base is more palatable. See In re Pearson, 494 F.2d 1399, 1405 (CCPA 1974) (“Attorney’s argument in a brief cannot take the place of evidence.”). We have already addressed Appellants unexpected results argument above. Conclusion of Law (i) The evidence of record supports the Examiner’s conclusion that Liversidge, DIH, and Bell-Huff suggest a “nanoparticulate sildenafil free base composition” as required by Claim 1? (ii) Appellants have not presented evidence of secondary considerations, that when weighed with the evidence of obviousness, are sufficient to support a conclusion of non-obviousness. F. and G. Obviousness Type Double Patenting The Examiner finds that “[b]oth applications claim compositions contemplating the presence of more than one active agent wherein the contemplated additional potential active agents in both applications substantially overlap in scope (e.g. claims 28-31 of copending '539 and claims 22-27of the instant application)” (Ans. 19). The Examiner finds that “it would have been obvious to modify the claimed method of making of both copending '064 and the instant application to prepare compositions comprising both sildenafil free base and glipizide” (Ans. 21). Appeal 2010-011978 Application 10/895,405 22 Appellants argue that the double patenting rejection over US 10/619,539 “is overcome by the terminal disclaimer filed herewith, and therefore should be withdrawn” (App. Br. 27). Appellants argue that “Appellants’ claims directed to a nanoparticulate sildenafil composition would not extend the right to exclude others from using a nanoparticulate glipizide composition in copending Application No. 10/701,064.” (App. Br. 28). We find that the Examiner has the better position. Regarding the rejection of US 10/619,539, we note that the terminal disclaimer was not approved (see Ans. 30). We therefore summarily affirm this rejection. We also do not find persuasive Appellants’ argument that the claims of US 10/701,064 drawn to a nanoparticulate combination of glipizide and sildenafil do not conflict with the instant claims. There are inventors in common and both applications are assigned to Elan Pharma. Appellants do not argue that there is a bar under 35 U.S.C. § 121. Claim 19 of US 10/701,064 encompasses the specific embodiment where the nanoparticulate composition comprises glipizide (an antidiabetic drug), a surface stabilizer and sildenafil. Compare this to instant claim 24, which may comprise a nanoparticulate composition with sildenafil free base, a stabilizer and antidiabetic agents. We agree with the Examiner “that the inclusion of sildenafil as an additional active agent is an obvious modification of the claims of copending ‘064, as evidenced by dependent claim 19 of copending ‘064, which explicitly recites sildenafil as a suitable additional active agent” (Ans. 31). Appeal 2010-011978 Application 10/895,405 23 New Ground of Rejection Under the provisions of 37 C.F.R. § 41.50(b), we enter the following new grounds of rejection. Claims 27-29 are rejected under 35 U.S.C. § 112, second paragraph as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicants regard as the invention. Findings of Fact 16. Rotella10 (J. Med. Chem.) teaches that “the more selective PDE5 inhibitor 14 demonstrated activity comparable to sildenafil” (Rotella (J. Med. Chem.) 1258, col. 2). 17. Rotella (J. Med. Chem.) teaches a structure for “compound 14” reproduced below: A structure for Compound 14 is shown above. 10 Rotella et al., N-3-Substituted Imidazoquinazolinones: Potent and Selective PDE5 Inhibitors as Potential Agents for Treatment of Erectile Dysfunction, 43 J. MED. CHEM. 1257-1263 (2000). Appeal 2010-011978 Application 10/895,405 24 18. Rotella11 (Nature Reviews) teaches a structure for “compound 14” in Figure 6, reproduced in part below: “Figure 6 . . . The phosphodiesterase 5 (PDE5) inhibitors compounds 14 and 15 (Bristol-Myers Squibb) are related to PDE inhibitors that were previously disclosed” (Rotella (Nature Reviews) 679). Principles of Law Because claims delineate the patentee's right to exclude, the patent statute requires that the scope of the claims be sufficiently definite to inform the public of the bounds of the protected invention, i.e., what subject matter is covered by the exclusive rights of the patent. Otherwise, competitors cannot avoid infringement, defeating the public notice function of patent claims. Halliburton Energy Services, Inc. v. M-I LLC, 514 F.3d 1244, 1249 (Fed. Cir. 2008). “Even if a claim term's definition can be reduced to words, the claim is still indefinite if a person of ordinary skill in the art cannot translate the definition into meaningfully precise claim scope.” Id. at 1251. Analysis Claim 27 is drawn to a number of different PDE5 inhibitors, including “compound 14”. Appellants’ Specification does not provide any 11 Rotella et al., Phosphodiesterase 5 Inhibitors: Current Status and Potential Applications, 1 NATURE REVIEWS 674-682 (2002). Appeal 2010-011978 Application 10/895,405 25 information regarding “compound 14”, so Appellants rely upon the prior art to define and describe this compound (see App. Br. 8-9). In particular, Appellants point to US 6,548,508 which recites “compound 14 from Rotella D P. J. Med. Chem. 2000, 43, 1257” (‘508, col. 5, ll. 38-39). However, while Rotella (J. Med. Chem.) identifies a molecule with one particular structure as a PDE5 inhibitor called “compound 14” (FF 16-17), Rotella (Nature Reviews) identifies a molecule with a different structure as a PDE5 inhibitor called “compound 14” (FF 18). Since Appellants’ Specification does not describe the structure or any other characteristics of “compound 14” and since there is no way to distinguish which of Rotella’s compound 14 molecules is meant by the Specification, the use of “compound 14” as a descriptor in claim 27 is vague and indefinite. The ordinary artisan would not have been able to determine which molecule was intended by the term “compound 14”. Appeal 2010-011978 Application 10/895,405 26 SUMMARY In summary, we reverse the rejection of claims 27-29 under 35 U.S.C. § 112, first paragraph as failing to comply with the written description requirement. We reverse the rejection of claims 27-29 under 35 U.S.C. § 112, first paragraph, as not enabled for the compositions disclosed in claim 27. We affirm the rejection of claim 1 under 35 U.S.C. § 103(a) as obvious over Straub and Bell-Huff. Pursuant to 37 C.F.R. § 41.37(c)(1)(vii)(2006), we also affirm the rejection of claims 2, 4, 6-21, 24, 26, and 30-39, as these claims were not argued separately. We affirm the rejection of claims 22, 23, 25, 28, and 29 under 35 U.S.C. § 103(a) as obvious over Straub, Bell-Huff, and Maggi. We affirm the rejection of claim 1 under 35 U.S.C. § 103(a) as obvious over Liversidge, Bell-Huff, and DIH. Pursuant to 37 C.F.R. § 41.37(c)(1)(vii)(2006), we also affirm the rejection of claims 2, 4, 6-21, and 33-39, as these claims were not argued separately. We affirm the provisional rejection of claims 1, 2, 4, 6-24, and 30-39 on the ground of nonstatutory obviousness-type double patenting over claims 1, 10, 11, 15-26, 31, and 41 of copending US Application 10/619,539 and Merck. We affirm the provisional rejection of claims 1, 2, 4, 6-22, 24, and 30- 39 on the ground of nonstatutory obviousness-type double patenting over claims 1-24 of copending US Application 10/701,064 and Merck (Ans. 20- 21). Appeal 2010-011978 Application 10/895,405 27 This decision also contains new grounds of rejection pursuant to 37 C.F.R. § 41.50(b) (effective September 13, 2004, 69 Fed. Reg. 49960 (August 12, 2004), 1286 Off. Gaz. Pat. Office 21 (September 7, 2004)). 37 C.F.R. § 41.50(b) provides “[a] new ground of rejection pursuant to this paragraph shall not be considered final for judicial review.” 37 C.F.R. § 41.50(b) also provides that the Appellants, WITHIN TWO MONTHS FROM THE DATE OF THE DECISION, must exercise one of the following two options with respect to the new ground of rejection to avoid termination of the appeal as to the rejected claims: (1) Reopen prosecution. Submit an appropriate amendment of the claims so rejected or new evidence relating to the claims so rejected, or both, and have the matter reconsidered by the Examiner, in which event the proceeding will be remanded to the Examiner. . . . (2) Request rehearing. Request that the proceeding be reheard under § 41.52 by the Board upon the same record. . . AFFIRMED-IN-PART, 37 C.F.R. § 41.50(b) alw ELAN DRUG DELIVERY, INC. C/O FOLEY & LARDNER 3000 K STREET, N.W. SUITE 500 WASHINGTON DC 20007-5109 Application/Control No. 10/895,405 Applicant(s)/Patent Under Reexamination Ryde, Tuula A. Notice of References Cited Examiner Alstrum Acevedo, James Art Unit 1600 Page 1 of 1 U.S. PATENT DOCUMENTS * Document Number Country Code-Number-Kind Code Date MM-YYYY Name Classification A US- B US- C US- D US- E US- F US- G US- H US- I US- J US- K US- L US- M US- FOREIGN PATENT DOCUMENTS * Document Number Country Code-Number-Kind Code Date MM-YYYY Country Name Classification N O P Q R S T NON-PATENT DOCUMENTS * Include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages) U Rotella et al., N-3 Substituted Imidazoquinazolinones: Potent and Selective PDE5 Inhibitors as Potential Agents for Treatment of Erectile Dysfunction, 43 J. Med. Chem. 1257-1263 (2000). V David P. Rotella, PHOSPHODIESTERASE 5 INHIBITORS: CURRENT STATUS AND POTENTIAL APPLICATIONS, 1 Nature 674-681 (2000). W X *A copy of this reference is not being furnished with this Office action. (See MPEP § 707.05(a).) Dates in MM-YYYY format are publication dates. Classifications may be US or foreign. U.S. Patent and Trademark Office PTO-892 (Rev. 01-2001) Notice of References Cited Part of Paper No. Delete Last PagelAdd A Page Expedited Articles N-3-Substituted Imidazoquinazolinones: Potent and Selective PDE5 Inhibitors as Potential Agents for Treatment of Erectile Dysfunction David P. Rotella,*,† Zhong Sun,† Yeheng Zhu,† John Krupinski,# Ronald Pongrac,# Laurie Seliger,# Diane Normandin,# and John E. Macor† Discovery Chemistry and Cardiovascular Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 5400, Princeton, New Jersey 08543-5400 Received February 23, 2000 Phosphodiesterase type 5 (PDE5) inhibitors with improved PDE isozyme selectivity relative to sildenafil may result in agents for the treatment of male erectile dysfunction (MED) with a lower incidence of PDE-associated adverse effects. This paper describes the discovery of 14, a PDE5 inhibitor with improved potency and selectivity in vitro compared to sildenafil. This compound shows activity in a functional assay of erectile function comparable to that of sildenafil. Introduction The utility of sildenafil (1, Viagra; Chart 1) as an efficacious, orally active agent for the treatment of male erectile dysfunction (MED)1 has created significant interest in the discovery of additional phosphodiesterase type 5 (PDE5) inhibitors.2 PDE5 is the primary cGMP- hydrolyzing enzyme activity present in the corpus cavernosum, the smooth muscle in the penis which helps control vascular tone. When a man is sexually stimu- lated, nitric oxide is released from the cavernosal nerve. This activates soluble guanylyl cyclase in the corpus cavernosum, causing an increase in intracellular cGMP, which is normally hydrolyzed by PDE5. Inhibition of PDE5 elevates levels of the cyclic nucleotide, leading to enhanced relaxation of smooth muscle, increased arterial inflow, venous congestion, and ultimately an erection. Despite the efficacy of 1 as a treatment for MED, there are notable drawbacks associated with its use. Clinically significant adverse effects such as nau- sea, headache, cutaneous flushing, and visual distur- bances have been noted, and their incidence is dose- dependent. Certain of these are thought to be due to nonspecific inhibition of other PDEs, specifically PDE1 and PDE6.3,4 Thus, the identification of potent and more selective PDE5 inhibitors is of primary interest. This paper describes the discovery of an N-3-(fluorobenzyl)- imidazoquinazolinone that is more potent and selective in vitro as a PDE5 inhibitor compared to sildenafil. This compound demonstrates activity comparable to 1 in a functional assay of erectile dysfunction using rabbit corpus cavernosum tissue strips. Results and Discussion Using the prototypical PDE5 inhibitor zaprinast (2; Chart 1)5 as a template, directed screening identified 3 (Chart 1) as a moderately active but nonselective lead (Table 1). The potency of compound 3 was improved 10- fold by incorporation of an N-methylpiperazinesulfona- mide in the pendant alkoxybenzene ring, leading to compound 7 (Scheme 1). This SAR observation was analogous to that described by Terrett et al.1b in the development of sildenafil. While this improved activity was encouraging, it was apparent that this modification did not enhance isozyme selectivity compared to sildena- fil (Table 1). In an attempt to improve the potency and selectivity of this series, modification of the imidazole ring was investigated. The synthesis of an N-3-benzyl derivative of 7 was carried out as shown in Scheme 2. Key to the synthesis of 11 was the selective formation of the imidazole ring in 9 that did not also lead to quinazoli- none formation. This was achieved by stirring the diamine intermediate derived from 8 in formic acid overnight at room temperature. The formyl group on the amine ortho to the primary amide which also resulted from this transformation was cleaved by brief * Corresponding author: David P. Rotella. Phone: 609-818-5398. Fax: 609-818-3450. E-mail: david.rotella@bms.com. † Discovery Chemistry. # Cardiovascular Drug Discovery. Chart 1 1257J. Med. Chem. 2000, 43, 1257-1263 10.1021/jm000081+ CCC: $19.00 © 2000 American Chemical Society Published on Web 03/18/2000 treatment with acid, leading to 9 in good overall yield from dinitro intermediate 8. Acylation of the aniline with acid chloride 12 gave piperazine 10, which without purification was cyclized using potassium tert-butoxide in refluxing tert-butyl alcohol to furnish 11. In vitro, 11 maintained PDE5 potency (relative to 7) and also substantially improved the selectivity profile of the series (Table 1). Specifically, 11 was 20-fold selective for PDE6, 3400-fold selective for PDE1, and 600-fold selective for PDE4. Compounds 7 and 11 were compared to 1 in a secondary in vitro assay to evaluate their functional effects on smooth muscle relaxation in rabbit cavernosal tissue strips.6,7 This model measures potentiation of the normal smooth muscle relaxation process and reflects the indirect effect that a PDE5 inhibitor exerts on the target tissue. It is important to note that administration of sildenafil does not directly result in an erection (vide supra), rather an external stimulus is required to initiate the cascade. The data in Table 2 indicate that the unsubstituted benzimidazole 7 exhibited a dose- related effect and was as efficacious as sildenafil as measured by the potentiation of relaxation enhance- ment. The N-3-benzyl derivative 11 was less active than both 1 and 7. We speculated that a contributing factor to this reduced activity was the significantly higher molecular weight of 11 (MW ) 572), compared to either 1 (MW ) 474) or 7 (MW ) 482). This may reduce diffusion of the compound into smooth muscle cells of the corpus cavernosum where the drug must act. Carboxamides offer an alternative, lower molecular weight handle for incorporation of potency-enhancing substituents in the alkoxybenzene moiety.8 Making use of this variation, along with further optimization of the N-3-benzyl substituent, led to the synthesis of compound 14 (Scheme 3). Benzimidazole 9b was coupled with 4-bromo-2-propoxybenzoic acid to furnish an intermedi- ate amide, which was cyclized to afford 13. Cyanide substitution, hydrolysis to the corresponding carboxylic acid, and amide formation afforded 14 in good yield. Amide 14 displayed enhanced PDE5 potency (IC50 ) 0.48 nM), compared to sildenafil and further improved the PDE selectivity profile of 11 (Table 1). Significant inhibition (PDE IC50 < 1 μM) of other PDEs is limited to PDE6. In this instance, compound 14 was 60-fold selective for PDE5, compared to less than 10-fold selective for sildenafil. Note that the improved selectiv- ity of 14 can be attributed to both an increase in PDE5 potency and a decrease in affinity for PDE6 (Table 1). Evaluation of 14 (MW ) 472) in rabbit corpus caver- nosum tissue clearly showed a positive dose-related effect and improved efficacy compared to the higher molecular weight sulfonamide 11. Compound 14 proved to be similar in efficacy to both 1 and 7 as measured by the increase in the relaxation integral relative to the control (Table 2). This information suggests that 14 is better able to penetrate cells in the target tissue, compared to 11, but also shows that the improved in vitro potency relative to sildenafil did not lead to a measurable increase in functional efficacy. Neverthe- less, the data in Table 2 indicate that this group of N-3- benzylbenzimidazoles is worthy of further study as potential agents for the treatment of MED. Conclusion In summary, we have identified a quinazolinone template that provides potent PDE5 inhibitors. Addition of a benzyl moiety at N-3 of this template confers substantial improvement in PDE selectivity and potency compared to sildenafil. This improved selectivity should translate into an improved PDE-related side effect profile in vivo, based on experience to date with sildena- fil. In a functional assay of erectile function, the more selective PDE5 inhibitor 14 demonstrated activity com- parable to sildenafil based on the ability of the com- pound to relax rabbit corpus cavernosum tissue. Addi- tional studies with this series of molecules will be reported in due course. Experimental Section General. NMR spectra were obtained at 400 MHz (1H) and 100 MHz (13C) on a Varian DRX-400 spectrometer. Chemical shifts are reported in ppm downfield from TMS as an internal standard. Thin-layer chromatography was carried out using 2.5 × 7.5-cm silica gel 60 (250 μM layer) plates with UV detection. Magnesium sulfate was employed to dry organic extracts prior to concentration by rotary evaporation. Flash chromatography was done using EM Science silica gel 60 (230-400 mesh). Standard solvents from EM Science were used as received. Anhydrous solvents from EM Science or Aldrich and all other commercially available reagents were used without further purification. Melting points were taken using a Thomas-Hoover MelTemp apparatus. Microanalysis was carried out by the Analytical Chemistry department at Bristol-Myers Squibb. Preparative HPLC was carried out on a Shimadzu LC8A system using a YMC ODS-A 30 × 250-mm column eluting with a 30-min linear gradient from 90% solvent A to 90% solvent B (solvent A: 90% water/10% MeOH with 0.1% TFA, solvent B: 90% MeOH/10% water with 0.1% TFA). Low-resolution mass spectra were recorded using an LC-MS system consisting of a Micromass ZMD mass spectrometer in electrospray (M + H) mode and a Shimadzu LC10AT HPLC using a YMC ODS-A 3 × 50-mm column using the same solvents as noted above in a 2-min linear gradient. 2-Amino-4-chloro-5-nitrobenzamide (4). 4-Chloroan- thranilic acid (10.0 g, 56.5 mmol) was dissolved at room temperature with stirring in 190 mL of distilled water containing 8.98 g (84.7 mmol) Na2CO3. When the 4-chloroan- thranilic acid was completely dissolved, a 20% w/v solution of phosgene in toluene (84 mL) was added dropwise via a dropping funnel over 45 min. The resulting suspension was stirred at room temperature overnight under nitrogen. The product was collected by filtration and washed well with water. The resulting gray-white solid was dried in a vacuum oven at 60 °C overnight to provide 7-chloro-1,4-dihydro-2H-3,1-ben- zoxazine-2,4-dione (10.3 g, 52.3 mmol, 93%): 1H NMR (DMSO- d6) δ 11.93 (br s, 1H), 7.17 (d, J ) 1.5 Hz, 1H), 7.28 (dd, J ) 1.6 and 8.2 Hz, 1H), 7.91 (d, J ) 8.2 Hz, 1H). A portion of this material (5.1 g, 25.9 mmol) was added in portions over 40 min to a cold (0 °C) solution of concentrated (96-98%) sulfuric acid (15 mL) and concentrated (70%) nitric Table 1. PDE5 IC50 and Selectivity Ratios for Other PDEsa IC50 ratio compd PDE5 IC50 (nM)b PDE1/5 PDE2/5 PDE3/5 PDE4/5 PDE6/5 1 1.6 ( 0.5 140 >104 3500 2600 8 3 44 ( 19 200 360 300 100 1 7 5.3 ( 0.6 90 1300 5900 1600 2 11 5.3 ( 1.1 3400 >104 8800 600 20 14 0.48 ( 0.1 >105 >105 >105 4200 60 a Enzyme sources: PDE1, bovine heart; PDE2, rat kidney; PDE3, human platelet; PDE4, rat kidney; PDE5, human platelet; PDE6, bovine retina. b All IC50 determinations are averages based on 3 determinations; PDE5 values are represented as IC50 ( SD for at least 3 independent experiments. 1258 Journal of Medicinal Chemistry, 2000, Vol. 43, No. 7 Rotella et al. acid (15 mL). The reaction was stirred at 0 °C for 1 h, then filtered through a sintered glass funnel. The filtrate was cautiously poured into crushed ice (250 g) to precipitate a yellow-tan solid. This solid was washed well with water and dried overnight in a vacuum oven (60 °C) to furnish 7-chloro- 1,4-dihydro-6-nitro-2H-3,1-benzoxazine-2,4-dione (3.09 g, 12.7 mmol, 49%): 1H NMR (DMSO-d6) δ 12.30 (br s, 1H), 7.28 (s, 1H), 8.53 (s, 1H). This product (6.5 g, 26.8 mmol) was suspended in glacial acetic acid (70 mL). Ammonium acetate (6.2 g, 80.6 mmol) was added, and the resulting mixture was heated to 100 °C with stirring for 3 h. After cooling to room temperature, the brown solution was poured into distilled water (200 mL) to precipitate a yellow solid which was collected by filtration and washed well with water and ether. This material was first air-dried, then dried overnight under high vacuum to provide 4 (4.9 g, 23.0 mmol, 86%): 1H NMR (DMSO-d6) δ 6.92 (s, 1H), 7.49 (br s, 1H), 7.89 (br s, 2H), 8.22 (br s, 1H), 8.53 (s, 1H). 7-Chloro-6-nitro-2-(2-propoxyphenyl)-4(3H)-quinazoli- none (5). Compound 4 (3.50 g, 16.3 mmol) was dissolved in pyridine at room temperature (1 mol equiv). o-Propoxybenzoyl chloride (4.51 g, 22.8 mmol) was partially dissolved in a small quantity (<10 mL) of DMF, and this mixture was added to the pyridine solution. The resulting brown solution was heated to 80 °C for 2.5 h. The reaction mixture was cooled to room temperature and poured into distilled water to precipitate a Scheme 1a a (a) Phosgene/PhCH3/aq Na2CO3, rt 18 h, 93%; (b) HNO3/H2SO4, 0 °C 1 h, 49%; (c) HOAc, NH4OAc, 100 °C 3 h, 86%; (d) 2-propoxybenzoyl chloride/DMF/pyridine, 80 °C 2.5 h, 93%; (e) NaOH/H2O2, aq EtOH, reflux 2 h, 89%; (f) (i) chlorosulfonic acid, 0 °C to rt 4 h, (ii) 4-methylpiperazine, CH2Cl2, rt 2 h, 85%; (g) 2 M NH3/EtOH, sealed tube, 130 °C overnight, 72%; (h) 40 psi H2, EtOH/aq HCl, 10% Pd-C, rt overnight, 59%; (i) formic acid, reflux 3 h, 93%. Scheme 2a a (a) H2SO4/KNO3, 40-145 °C, 45%; (b) (i) (COCl)2, CH2Cl2, cat. DMF, 1 h, (ii) NH4OH, acetone, 0 °C 45 min, 75%; (c) (X)-benzylamine, THF, Et3N, reflux 1-2 h, 81-84%; (d) (i) 25 psi H2, PtO2, MeOH, 3-5 h; (ii) formic acid, rt overnight, (iii) 10% aq HCl/EtOH, rt 3 h, 90-93%; (e) 5-[(4-methylpiperazinyl)sulfonyl]-2-propoxybenzoyl chloride, pyridine/DMF, 75 °C 1-2 h; (f) tBuOK-tBuOH, reflux 2 h, 39% (two steps); (g) propyl iodide, K2CO3/DMF, rt overnight; (h) HSO3Cl/SOCl2, 0 °C 30 min, 28% net; (i) 4-methylpiperazine, triethylamine, CH2Cl2, 0 °C 1.5 h, 100%; (j) LiOH, THF-H2O, reflux 16 h, 96%; (k) (COCl)2, CH2Cl2, cat. DMF, 2 h. Table 2. Rabbit Corpus Cavernosum Functional Assay % control relaxation integral compd 30 nMa 300 nMa 1 150 ( 20 220 ( 25 7 140 ( 10 210 ( 30 11 120 ( 10 150 ( 13 14 140 ( 12 190 ( 32 a Control (untreated) response ) 100%. N-3-Substituted Imidazoquinazolinones Journal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1259 brown solid. This suspension was stirred at room temperature overnight. In the morning, the solid was collected by filtration and washed with water, 10% HCl, and then ether. The product (4-chloro-2-[(2-propoxybenzoyl)amino]-5-nitrobenzamide) was obtained in 93% yield (5.70 g, 15.2 mmol) after drying in a vacuum oven: mp 177-178 °C; 1H NMR (DMSO-d6) δ 9.04 (s, 1H), 8.55 (s, 2H), 8.05 (s, 1H), 7.88 (d, J ) 6.5 Hz, 1H), 7.59 (apparent t, J ) 6.2 Hz, 1H), 7.23 (d, J ) 6.5 Hz, 1H), 7.10 (apparent t, J ) 6.2 Hz, 1H), 4.19 (t, J ) 7.2 Hz, 2H), 1.84 (m, 2H), 0.92 (t, J ) 7.4 Hz, 3H). This material was suspended in absolute ethanol (15 mL ethanol), and water was added (7 mL). Sodium hydroxide (0.73 g, 18.2 mmol) was then added, followed by 0.86 mL (0.26 g, 7.6 mmol) of 30% (w/v) aqueous hydrogen peroxide. The reaction mixture was then heated to reflux, and the starting material gradually dissolved. When the starting material was consumed as determined by TLC analysis (generally in less than 2 h), the reaction was cooled to room temperature and concentrated by rotary evaporation to furnish a yellow-brown solid which was washed with water and triturated with ether to provide an 89% yield (4.38 g, 12.2 mmol) of 5: mp 178-181 °C; 1H NMR (DMSO-d6) δ 8.80 (s, 1H), 8.59 (apparent d, J ) 8 Hz, 1H), 7.91 (s, 1H), 7.57 (apparent t, J ) 8 Hz, 1H), 7.19 (apparent t, J ) 7.4 Hz, 1H), 7.09 (d, J ) 8 Hz, 1H), 4.24 (t, J ) 6.5 Hz, 2H), 2.04 (m, 2H), 1.18 (t, J ) 7.4 Hz, 3H). 1-[[3-(7-Chloro-3,4-dihydro-6-nitro-4-oxo-2-quinazoli- nyl)propoxyphenyl]sulfonyl]-4-methylpiperazine (6). Chlo- rosulfonic acid (10 mL) was cooled to 0 °C in ice under nitrogen. Compound 5 (0.81 g, 2.3 mmol) was added portionwise over 20-30 min. The reaction was stirred at 0 °C for 5 h then, very cautiously, poured slowly into crushed ice. The resulting yellow precipitate was collected by filtration, washed thoroughly with water and sucked dry with a water aspirator. This material was used without further purification for sulfonamide forma- tion. The resulting sulfonyl chloride was partially dissolved in 20 mL of methylene chloride/2 mL of THF. Triethylamine (0.31 g, 3.04 mmol, 423 μL) was added. This was followed by 0.24 g (264 μL, 2.39 mmol) of 4-methylpiperazine. The reaction mixture was stirred at room temperature for 2 h then diluted with additional methylene chloride and washed twice with water, dried over magnesium sulfate and concentrated to furnish the product as a yellow solid in 72% yield (0.86 g, 1.66 mmol): mp 225-228 °C; LRMS [MH+] 522; 1H NMR (CDCl3) δ 8.94 (d, 1H, J ) 2.4 Hz), 8.79 (s, 1H), 7.98 (s, 1H), 7.92 (dd, 1H, J ) 2.4, 8.7 Hz), 7.22 (d, 1 H, J ) 8.7 Hz), 4.32 (t, 2H, J ) 6.6 Hz), 3.08-3.12 (m, 4H), 2.43-2.56 (m, 4H), 2.28 (s, 3H), 2.05-2.10 (m, 2H), 1.40 (t, 2H, J ) 7.2 Hz), 1.19 (t, 3H, J ) 7.2 Hz). 1-[[3-(7,8-Dihydro-8-oxo-1H-imidazo[4,5-g]quinazolin- 6-yl)-4-propoxyphenyl]sulfonyl]-4-methylpiperazine (7). Compound 6 (0.65 g, 1.24 mmol) was suspended in equal volumes of absolute ethanol and 28% aqueous ammonium hydroxide in a pressure bottle (total volume 25 mL) with a stirring bar. After the bottled was tightly sealed, the contents were heated at 140 °C overnight. The reaction mixture was cooled to room temperature, and the resulting suspension was diluted with water. The resulting mixture was filtered to afford a bright yellow solid. This solid was washed with water, ethanol, and ether to provide 1-[[3-(7-amino-3,4-dihydro-6- nitro-4-oxo-2-quinazolinyl)-4-propoxyphenyl]sulfonyl]-4-meth- ylpiperazine (0.44 g, 0.87 mmol, 70% yield): mp 270-271 °C; LRMS [MH+] 503; 1H NMR (DMSO-d6) δ 8.75 (s, 1H), 7.94 (d, 1H, J ) 2.3 Hz), 7.86 (dd, 1h, J ) 2.3, 8.6 Hz), 7.72 (s, 1H), 7.41 (d, 1H, J ) 8.7 Hz), 7.12 (s, 1H), 4.13 (t, 2H, J ) 6.2 Hz), 2.90 (br s, 4H), 2.37 (br s, 4H), 2.15 (s, 3H), 1.71-1.77 (m, 2H), 0.95 (t, 3H, J ) 6.2 Hz). This material was partially dissolved in 5 mL of 10% aqueous HCl and added to a suspension of 10% palladium on charcoal (50 wt %) in absolute ethanol (20 mL) in a Parr bottle. The mixture was hydrogenated on a Parr shaker at room temperature under 40 psi H2 overnight. The suspension was filtered through Celite and the cake washed well with ethanol. The filtrate was evaporated to provide 1-[[3-(6,7-diamino-3,4- dihydro-4-oxo-2-quinazolinyl)-4-propoxyphenyl]sulfonyl]-4-me- thylpiperazine as the hydrochloride salt (0.21 g, 0.44 mmol, 50% yield). This material was used without further purifica- tion: LRMS [MH+] 473; 1H NMR (CD3OD) δ 8.22 (s, 1H), 8.18 (d, 1H, J ) 8.6 Hz), 8.00 (s, 1H), 7.58 (d, 1H, J ) 8.6 Hz), 7.15 (s, 1H), 4.24 (t, 2H, J ) 6.2 Hz), 3.94 (br d, 4H, J ) 8.2 Hz, D2O exchangeable), 3.24 (m, 4H), 2.85-3.05 (m, 7H), 1.80- 1.93 (m, 2H), 1.00 (t, 3H, J ) 6.2 Hz). This diamine (0.20 g, 0.39 mmol) was dissolved in 10 mL of concentrated formic acid and heated to reflux under nitrogen. The reaction was followed by HPLC, and generally conversion to product was complete in 2 h or less. The reaction was cooled to room temperature and formic acid was removed in vacuo. Residual water was azeotropically removed with ethanol leaving a light brown to reddish brown solid. The solid was dissolved in 10% aqueous HCl and washed with three portions of ethyl acetate. The pH of the water layer was adjusted to 12 with sodium hydroxide solution and extracted five times with ethyl acetate. The collected organic extracts were washed twice with brine, dried and concentrated. Further purification was accomplished by dissolving this material in 20 mL of EtOAc, which was cooled in ice before HCl gas was passed through the solution to deposit fine tan needles of the hydrochloride salt of the product (0.14 g, 0.26 mmol, 66% yield): mp (free base) 164-167 °C; LRMS [MH+] 483; 1H NMR (CD3OD) δ 9.59 (s, 1H), 8.66 (s, 1H), 8.22 (d, 1H, J ) 2.4 Hz), 8.17 (s, 1H), 7.96 (dd, 1H, J ) 2.4, 8.7 Hz), 7.40 (d, 1H, J ) 8.7 Hz), 4.16 (t, 2H, J ) 6.3 Hz), 3.87 (br d, 2H, J ) 12.8 Hz), 3.50 (br d, 2H, J ) 12.8 Hz), 3.1-3.2 (m, partially obscured by MeOH, 2H), 2.8-2.92 (m, 5H), 1.75-1.88 (m, 2H), 0.95 (t, 3H, J ) 7.4 Hz). Anal. Calcd for C23H27ClN6O4S: C, 53.22; H, 5.24; Cl, 6.83; N, 16.19; S, 6.18. Found: C, 53.20; H, 5.25; Cl, 6.79; N, 16.14. 2,4-Dinitro-5-chlorobenzoic Acid. 3-Chlorobenzoic acid (12.5 g, 80 mmol) was dissolved in 145 mL of concentrated sulfuric acid with stirring, while warming to 40 °C. Potassium nitrate (8.0 g, 78 mmol) was added in divided portions over 30 min. The reaction mixture was then warmed to 100 °C and an additional 14 g of potassium nitrate was added over 20 min. The reaction mixture was warmed to 145 °C and held at this temperature for 15 min. The reaction was cooled to room temperature and poured into 1 kg of ice to precipitate a faintly yellow solid. This material was collected by filtration and washed with water. The resulting solid was then suspended in 500 mL of distilled water and stirred at room temperature for 45 min. The undissolved solid was collected by filtration and dried under high vacuum to obtain 8.9 g (36%) yield of product as a faintly yellow solid: 1H NMR (acetone-d6) δ 8.76 (s, 1H), 8.26, (s, 1H). 2,4-Dinitro-5-chlorobenzamide. 2,4-Dinitro-5-chloroben- Scheme 3a a (a) 2-Propoxy-4-bromobenzoic acid, HOBt, EDAC, cat. DMAP, DMF, 4 h; (b) tBuOK, tBuOH, reflux 2 h, 82%; (c) CuCN, N-methylpyrrolidinone, reflux 18 h, 91%; (d) NaOH, EtOH, reflux 5 h, 75%; (e) NH3/THF, EDAC, HOBt, cat. DMAP, pyridine, 82%. 1260 Journal of Medicinal Chemistry, 2000, Vol. 43, No. 7 Rotella et al. zoic acid (7.00 g, 28.4 mmol) was suspended in 40 mL of thionyl chloride containing 3 drops of DMF. The suspension was warmed to reflux for 4 h. After cooling to room temperature, solvent was removed by rotary evaporation leaving a golden yellow liquid. This was diluted with 30 mL of acetone and added dropwise over 20 min to a 0 °C solution of 20 mL of concentrated ammonium hydroxide. The reaction was stirred at 0 °C for 30 min, then poured into 250 g of ice. The yellow orange precipitate was collected by filtration and washed well with water. The solid was dried first by water aspirator then under high vacuum to furnish 5.9 g (24 mmol, 84% yield) of product: 1H NMR (acetone-d6) δ 8.75 (s, 1H), 8.25 (s, 1H); mp 201-203 °C. General Procedure for Preparation of 5-Benzylamino- 2,4-dinitrobenzamides. 2,4-Dinitro-5-chlorobenzamide (2.00- 14.25 mmol) was suspended in 15-70 mL of THF and 1.2 equiv of triethylamine was added, followed by 1.2 equiv of the appropriate benzylamine. The reaction was heated to reflux until TLC indicated starting material had been consumed. The cooled reaction mixture was filtered and the filtrate was concentrated in vacuo leaving a solid which was triturated with ether. This solid was collected by filtration, washed with ether and dried affording the product. 5-[(Phenylmethyl)amino]-2,4-dinitrobenzamide (8a): 95% yield; mp 143-146 °C; 1H NMR (acetone) δ 9.21 (br s, 1H), 8.90 (s, 1H), 7.31-7.49 (m, 5H), 7.13 (s, 1H), 7.02 (br s, 1H), 4.90 (d, 2H, J ) 6 Hz). 5-[[(4-Fluorophenyl)methyl]amino]-2,4-dinitrobenza- mide (8b): 81% yield; mp 178-180 °C; 1H NMR (DMSO) δ 9.44 (br t 1H, exchangeable with D2O), 8.72 (s, 1H), 8.35 (br s, 2H, exchangeable with D2O), 7.40-7.44 (m, 2H), 7.15-7.21 (m, 2H), 7.02 (s, 1H), 4.75 (d, 2H, J ) 6.3 Hz). General Procedure for Synthesis of 5-Amino-1-[(phe- nylmethyl)amino]-1H-benzimidazole-6-carboxamides. The appropriate 5-benzylamino-2,4-dinitrobenzamide (8a or 8b from above) (0.50-3.5 g) was partially dissolved in methanol (20-200 mL) containing 25 wt % platinum oxide. Hydrogen was introduced using a balloon and the flask was evacuated and filled several times before leaving the suspension stirring under an atmosphere of hydrogen at room temperature. The mixture was stirred until HPLC analysis showed consumption of starting material and conversion to the desired diamine product (typically 5 h). The suspension was filtered through Celite and the filter cake was washed with methanol. The filtrate was concentrated to furnish the crude diamine. This material was then dissolved in concentrated (96%) formic acid and stirred at room temperature overnight. Formic acid was removed at room temperature under vacuum, leaving a brown solid. This material was dissolved in absolute ethanol (10-50 mL) and 10% aqueous HCl (3-10 mL) and stirred at room temperature for 3 h. Solvent was removed by rotary evapora- tion to furnish the product as the hydrochloride salt. This material was sufficiently pure for further transformation. The yield stated is the net for this three-step sequence. 5-Amino-1-[(phenylmethyl)amino]-1H-benzimidazole- 6-carboxamide (9a): 90% yield; LRMS [MH+] 267; 1H NMR (CD3OD) δ 9.68 (s, 1H), 8.55 (s, 1H), 7.98 (s, 1H), 7.40-7.52 (m, 5H), 5.81 (s, 2H). 5-Amino-1-[[(4-fluorophenyl)methyl]amino]-1H-benz- imidazole-6-carboxamide (9b): 93% yield; LRMS [MH+] 286; 1H NMR (CD3OD) δ 9,73 (s, 1H), 8.64 (s, 1H), 7.60-7.68 (m, 2H), 7.19 (apparent t, 2H, J ) 8.5 Hz), 5.83 (s, 2H). 5-[(4-Methyl-4-piperazinyl)sulfonyl]-2-propoxybenzo- ic Acid, Lithium Salt. To a solution of methyl salicyclate (25.00 g, 0.16 mol) in 200 mL of DMF were added potassium carbonate (34.00 g, 0.25 mol) and 1-iodopropane (84.00 g, 0.49 mol). The mixture was stirred at room temperature for 24 h. The reaction was diluted with 400 mL of water and extracted with 5 × 100 mL of ether. The combined organic extracts were washed twice with brine, dried and concentrated to give a faintly yellow liquid that contained the product, methyl-2- propoxybenzoic acid and excess 1-iodopropane. This mixture was added dropwise at 0 °C to a mixture of 35 mL of chlorosulfonic acid and 10 mL of thionyl chloride over 30 min. The dark red reaction was allowed to slowly warm to room temperature overnight. The mixture was cautiously poured over 1 kg of ice and stirred to deposit a yellow solid that was recrystallized from cyclohexane to furnish 13.00 g (0.045 mol, 28% yield) of the corresponding sulfonyl chloride: mp 58-59 °C; 13C NMR (CDCl3) δ 164.7, 163.9, 135.6, 132.7, 131.8, 121.5, 113.6, 71.6, 52.9, 22.6, 10.7. 1.3 g of this compound (6.23 mmol) was dissolved in 20 mL of methylene chloride and cooled to 0 °C in ice; 0.82 g (8.10 mmol) of triethylamine was added, followed by 0.69 g (6.86 mmol) of 4-methylpiperazine. The reaction was stirred in ice for 1.5 h, then diluted with 50 mL of methylene chloride and washed twice with water and dried. The organic phase was concentrated in vacuo leaving a clear colorless oil which partially solidified under vacuum to give 2.21 g (6.23 mmol, 100% yield) of the desired sulfonamide: 1H NMR (CDCl3) δ 8.15 (d, 1H, J ) 2.5 Hz), 7.81 (dd, 1H, J ) 2.5, 8.9 Hz), 7.05 (d, 1H, J ) 8.9 Hz), 4.07 (t, 2H, J ) 6.4 Hz), 3.90 (s, 3H), 3.03 (br apparent s, 4H), 2.47 (apparent t, 4H, J ) 4.8 Hz), 2.26 (s, 3H), 1.86-1.91 (m, 2H), 1.09 (t, 3H, J ) 7.3 Hz). This ester was dissolved in 45 mL of THF with 5 mL of water and 0.27 g (6.23 mmol) of lithium hydroxide monohy- drate. The solution was heated to reflux overnight and in the morning, solvents were removed in vacuo leaving the product as a white solid (2.17 g, 6.23 mmol, 100%): 1H NMR (D2O) δ 7.62 (dd, 1H, J ) 2.5, 8.7 Hz), 7.57 (d, 1H, 2.5 Hz), 7.09 (d, 1H, J ) 8.7 Hz), 3.97 (t, 2H, J ) 6.5 Hz), 2.89 (br s, 4H), 2.38 (br, s, 4H), 2.06 (s, 3H), 1.60-1.69 (m, 2H), 0.84 (t, 3H, J ) 7.5 Hz). 1-[[3-[7,8-Dihydro-8-oxo-1-(phenylmethyl)-1H-imidazo- [4,5-g]quinazolin-6-yl]-4-propoxyphenyl]sulfonyl]-4-me- thylpiperazine (11). 5-[(4-Methyl-4-piperazinyl)sulfonyl]-2- propoxybenzoic acid, lithium salt (1.4 g, 3.9 mmol) was dissolved in 20 mL of methylene chloride containing 3 drops of DMF at 0 °C. Oxalyl chloride (0.70 g, 5.11 mmol, 0.48 mL) was added dropwise over 20 min. The cold bath was removed and the reaction stirred at room temperature for 2 h. Solvents were removed on a rotary evaporator and the residue was suspended in 25 mL of pyridine and 2 mL of DMF. To this was added 9a (0.91 g, 3 mmol) and the reaction was heated to 75 °C for 30 min. The reaction was poured into cold water and extracted with methylene chloride (4 × 25 mL). The organic extract was washed with water and brine and dried, then concentrated in vacuo to afford a dark brown semisolid (1.2 g) that was used without further purification for the next reaction. This crude material was suspended in 15 mL of dry tert- butyl alcohol and 4.4 mL of KOtBu (1.0 M in tBuOH) was added. The solution was heated to reflux under argon for 45 min. Water (25 mL) was added to the reaction to precipitate a brown solid that was collected by filtration, washed with water and dried to furnish 0.45 g (0.78 mmol, 39% yield) of product: mp 125-127 °C; 1H NMR (CD3OD) δ 8.63 (s, 1H), 8.32 (s, 1H), 8.25 (d, 1H, J ) 2.3 Hz), 7.90 (d, 1H, J ) 2.3 Hz), 7.88 (d, 1H, J ) 2.4 Hz), 7.29-7.39 (m, 5H), 5.60 (d, 2H, J ) 13 Hz), 4.19 (t, 2H, J ) 6.3 Hz), 3.07 (br s, 4H), 2.51 (m, 4H), 1.81-1.90 (m, 2H), 1.03 (t, 3H, J ) 7.4 Hz). An analytical sample was obtained following preparative HPLC. Anal. Calcd for C30H32N6O4S‚1.3H2O: C, 51.73; H, 4.97; N, 11.10; S, 4.24. Found: C, 51.52; H, 5.00; N, 11.09; S, 4.52. 1-[3-[1-[(4-Fluorophenyl)methyl]-7,8-dihydro-8-oxo-1H- imidazo[4,5-g]quinazolin-6-yl]-4-propoxyphenyl]carbox- amide (14). 2-Propoxy-4-bromobenzoic acid (2.60 g, 10.0 mmol) was dissolved in 25 mL of pyridine. To this were added 9b (3.20 g, 10 mmol), EDAC (2.30 g, 12 mmol), DMAP (0.18 g 1.5 mmol) and HOBt (1.84 g, 12 mmol). The reaction was stirred at room temperature for 2 h. Most of the pyridine was removed in vacuo and 50 mL of water was added to precipitate the product which was collected by filtration, washed with water and dried to afford 4.57 g of material which was used without further purification. This material was cyclized as described above for the preparation of 11 to furnish compound 13 as a beige solid in 82% yield (3.47 g): mp 295-297 °C; LCMS [MH+] 508; HPLC (YMC S5 ODS 4.6 × 50-mm column, N-3-Substituted Imidazoquinazolinones Journal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1261 4-min gradient of 0% B to 100% B, 4 mL/min flow, solvent A: 10% MeOH-90% H2O-0.2% H3PO4, solvent B: 90% MeOH- 10% H2O-0.2% H3PO4) retention time 4.19 min (94% pure). Without further purification, 1.50 g of this material was dissolved in 15 mL of N-methyl-2-pyrrolidinone containing copper(I) cyanide (0.53 g, 5.9 mmol). The reaction was heated to reflux overnight, then cooled to room temperature and poured into 100 mL of 2 N aqueous ammonia solution. The solid that precipitated was collected by filtration and washed first with 2 N aqueous ammonia then water and dried in a vacuum oven at 45 °C overnight to afford the product 1-(4- fluorophenylmethyl)-6-(4-cyano-2-propoxyphenyl)-1H-imidazo- [4,5-g]quinazolin-8(7H)-one as a yellow solid (1.20 g, 2.69 mmol, 91% yield): LC-MS [MH+] 454; HPLC (YMC S5 ODS 4.6 × 50-mm column, 4-min gradient of 0% B to 100% B, 4 mL/min flow, solvent A: 10% MeOH-90% H2O-0.2% H3PO4, solvent B: 90% MeOH-10% H2O-0.2% H3PO4) retention time 3.65 min. This compound (0.50 g, 1.10 mmol) was added to 10 mL of absolute ethanol containing 10 mL of 10% (w/v) NaOH. The reaction mixture was heated to reflux for 5 h then cooled to room temperature. Undissolved solids were removed by filtra- tion and ethanol was removed under reduced pressure. Water (100 mL) was added and the solution was acidified with 10% HCl to pH 2-3. The resulting precipitate was collected by filtration and washed with distilled water, then dried under vacuum at 45 °C overnight to provide the corresponding carboxylic acid in 75% yield (0.39 g, 0.83 mmol) as a sand- colored solid: mp 295 °C; LC-MS [MH+] 473. This carboxylic acid (0.083 g, 0.18 mmol) was dissolved in 3 mL of pyridine containing 0.040 g (0.22 mmol) of EDAC- HCl, 0.032 g (0.22 mmol) of HOBt, ammonia (0.42 mL of a 0.5 M solution in dioxane) and 0.004 g (0.03 mmol) of (dimethy- lamino)pyridine. The reaction was stirred at room temperature for 2-5 h. Most of the pyridine was removed under reduced pressure and the residue was poured into water. The precipi- tated solid was collected by filtration and washed with water then dried. This material was added to 10 mL of MeOH, to which was added 10 drops of 10% aqueous HCl. The undis- solved solids were removed by filtration and the filtrate was concentrated. This solid was triturated with ether then recrystallized from MeOH to furnish 0.058 g of 14 (82% yield) as a light tan solid: mp 204-206 °C; LRMS [MH+] 473; 1H NMR (DMSO) δ 9.29 (br s, 1H), 8.48 (s, 1H), 8.27 (s, 1H), 8.00- 8.12 (m, 3H), 7.50-7.60 (m, 2H), 7.37 (s, 1H), 7.22-7.27 (m, 3H), 5.78 (s, 2H), 4.10 (t, 2H, J ) 6.3 Hz), 1.67-1.76 (m, 2H), 0.94 (t, J ) 7.3 Hz). Anal. Calcd for C26H22N5O3F‚HCl‚H2O: C, 55.57; H, 5.20; N, 12.46; Cl, 6.31. Found: C, 55.62; H, 5.18; N, 12.61; Cl, 6.45. PDE Enzyme Assay Procedure. PDE activity assay: Enzymatic activity was assayed using a commercially available PDE scintillation proximity (SPA) assay kit with either [3H]- cGMP or [3H]cAMP as the substrate depending on the PDE of interest (Amersham product #TRKQ 7090 for cAMP kit, #TRKQ 7100 for cGMP kit). The manufacturer’s protocol was followed explicitly except that the reactions were carried out at room temperature and 3 mM nonradioactive cyclic nucle- otide was included in the suspension of SPA beads to stop the synthesis of additional radioactive products. The radioactive product of the reaction, [3H]nucleotide monophosphate, pref- erentially bound to the SPA beads, excited the scintillant embedded in the beads, and was quantified on a Packard TopCount liquid scintillation counter. Nonspecific binding to the beads (the assay blank) was quantified by adding a 10000- fold excess of nonradioactive substrate to the reaction mixture prior to the addition of enzyme to prevent the synthesis of radioactive product. The assay blank was subtracted from the activity of the enzyme measured using the SPA kit as described above. The activity in samples that received test compound was calculated as a percent of the control activity measured in samples that only received the vehicle. Enzyme preparations: For either PDE3 or PDE5 activity, the enzyme source was sonicated human platelet homogenates prepared by the method of Seiler et al. (Seiler, S.; Gillespie, E.; Arnold, A. J.; Brassard, C. L.; Meanwell, N. A.; Fleming, J. S. Imidazoquinoline derivatives: potent inhibitors of platelet cAMP phosphodiesterase which elevate cAMP levels and activate protein kinase in platelets. Thromb. Res. 1991, 62, 31-42). PDE5 accounted for approximately 90% of the [3H]- cGMP hydrolytic activity in the homogenates and PDE3 accounted for over 80% of the [3H]cAMP hydrolytic activity. PDE1 from bovine heart was purchased from Sigma and assayed with 1 μM calmodulin and 4 mM calcium present in the reaction buffer and [3H]cGMP as the substrate. PDE2 and PDE4 were purified from rat kidney cytosol by Mono-Q anion- exchange chromatography as described (Hoey, M.; Houslay, M. D. Identification and selective inhibition of four distinct soluble forms of cyclic nucleotide phosphodiesterase activity from kidney. Biochem. Pharmacol. 1990, 40, 193-202). The substrate for both of these isoforms was [3H]cAMP. Nonra- dioactive cGMP (1 μM) was also included in the reaction buffer for the PDE2 assay because the cAMP hydrolytic activity of this isoform is stimulated by cGMP. Rod outer segment membranes were purified from bovine retinas as described (Fung, B. K.-K.; Stryer, L. Photolyzed rhodopsin catalyzes the exchange of GTP for bound GDP in retinal rod outer sefments. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 2500-2504). PDE6 was stripped from the rod outer segment membranes by hypotonic washes and trypsin-activated prior to the assay as described (Berger, A. L.; Cerione, R. A.; Erickson, J. W. Real time conformational changes in the retinal phosphodiesterase γ subunit monitored by resonance energy transfer. J. Biol. Chem. 1997, 272, 2714-2721). Data analysis: Each data set was fit to a curve for inhibition at a single site using the nonlinear regression analysis in the Activity Base/XLFit software package, and IC50 values were calculated by this analysis. Selectivity values were calculated as the ratio of the IC50 value for inhibition of one PDE divided by the IC50 value for inhibition of PDE5. Rabbit Corpus Cavernosum Strip Assay. Physiologi- cal salt solutions: A bicarbonate-buffered salt solution (PSS) was used, containing (in mM): 118.4 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.9 CaCl2, 25.0 NaHCO3, and 10.1 D-glucose. The solution additionally contained 3 μM indometha- cin, 1 μM atropine, and 5 μM guanethidine. Concentrated stock solutions (10 mM) of test compounds were prepared and serially diluted to the appropriate concentrations in DMSO. Appropriate solvent/time controls were run in parallel. Tissue preparation: Adult male New Zealand white rabbits weighing 3.4-3.7 kg were sacrificed by intravenous Nembutal injection. The entire penis (up to pelvic bone) was quickly removed and placed in ice-cold aerated PSS. The skin and connective tissue were carefully removed. The corpus spongiosum was cut from the groove under the corpus caver- nosum. The tunica albuginea was dissected from the corpus cavernosum. The corpus cavernosum was carefully cut along the midline and connective tissue was cleaned from the “septal” region. The two corpora were longitudinally cut in half; each of these strips was then cut in half along the transverse axis, resulting in eight strips approximately 2 × 5 mm. Each strip was individually mounted for isometric force recording using silk suture in 10-mL tissue baths. One end was secured between the two platinum plates of a field-stimulating elec- trode, which in turn was connected to a micrometer for control of tissue length. The field-stimulating electrode was attached to a Stimu-splitter and Grass stimulator. The other end of the suture was connected to a Grass FT.03 force displacement transducer. The strips were bathed in PSS maintained at 37 °C and bubbled with 95% O2 and 5% CO2. Data were simultaneously recorded on a Grass recorder and a Biopac System which additionally allowed measurement of the inte- gral at each frequency. A preload tension of 1 g was applied to each strip. Following the equilibration period, the strips were contracted with 3 μM PE until a steady state level of force was attained. For the first frequency/response curve, 3 μM DMSO (solvent control) was added to each strip for 10 min. A frequency/response curve of the following parameters was then performed: 24 V in the form of square wave pulses of 1262 Journal of Medicinal Chemistry, 2000, Vol. 43, No. 7 Rotella et al. 0.2-ms duration delivered as 10-s train and a 4-min interval between stimuli. A frequency of 32 Hz was chosen to measure the effects of test compounds based on the signal-to-noise response. After a 4-min recovery at 32 Hz, the strips were washed extensively and allowed at least 50 min to recover during which time tension adjustments and several washes were done. The frequency/response curve was repeated in the presence of test compound. Following another recovery period, a third frequency/response curve was performed in the pres- ence of a higher concentration of test compound. Data analysis: All force determinations were made assum- ing that the lowest level of force following a washout period defined 100% relaxation and the phenylephrine contraction prior to DMSO or test compound addition was 0% relaxation. The direct relaxation induced by test compound, measured after the 10-min incubation period, was calculated relative to these values. The peak relaxation responses at 32 Hz was also measured relative to these values. Values were then normal- ized to the maximum peak relaxation attained during the control frequency response curve. The integral for the area under the curve at 32 Hz was determined for a set time period (3.66 s from immediately prior to stimulation). The integral for the relaxation response was then calculated as the differ- ence between the integral for the time period immediately prior to the start of stimulation and that for the frequency. All responses were normalized to the maximum attained during the first frequency/response curve. Each data set was fit to a sigmoidal dose-response curve by nonlinear regression using the GraphPad Prism software package. References (1) Drugs Future 1997, 22, 138-143. (b) Terrett, N. K.; Bell, A. S.; Brown, D.; Ellis, P. Sildenafil (Viagra), a potent and selective inhibitor of type 5 cGMP phosphodiesterase with utility for the treatment of male erectile dysfunction. Bioorg. Med. Chem. Lett. 1996, 6, 1819-1824. (c) Boolell, M.; Allen, M. J.; Ballard, S. A.; Gepi-Attee, S.; Muirhead, G. J.; Naylor, A. M.; Osterloh, I. H.; Gingell, C. Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int. J. Urol. Res. 1996, 8, 47-52. (2) Eardley, I. The role of phosphodiesterase inhibitors in impotence. Exp. Opin. Invest. Drugs 1997, 6, 1803-1810. (b) Garcia-Reboll, L.; Mulhall, J. P.; Goldstein, I. Drugs for the treatment of impotence. Drugs Aging 1997, 11, 140-151. (c) Truss, M. C.; Stief, C. G. Phosphodiesterase inhibitors in the treatment of erectile dysfunction. Drugs Today 1998, 34, 805-812. (3) Beavo, J. A. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol. Rev. 1995, 75, 725- 748. (4) Scrip 1997, October 9. (5) Lugnier, C.; Schoeffter, P.; Le Bec, A.; Strouther, E.; Stoclet, J. C. Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta. Biochem. Pharmacol. 1986, 35, 1743-1751. (6) Electrical field stimulation causes the release of nitric oxide (NO) from endogenous nerve terminals, resulting in a frequency- dependent relaxation of phenylephrine-precontracted strips of rabbit corpus cavernosum in the absence of a PDE5 inhibitor. Addition of a PDE5 inhibitor can potentiate the electrical field- stimulated relaxation. This potentiation is expressed as an increase in the relaxation integral at 32 Hz, with the maximum relaxation integral in the presence of vehicle alone defined as 100%. Sildenafil is used as a positive control in each experiment. The method used herein is a modification of that employed in ref 7b. (7) Stief, C. G.; Uckert, S.; Becker, A. J.; Truss, M. C.; Jonas, U. The effect of specific phosphodiesterase (PDE) inhibitors on human and rabbit cavernous tissue in vitro and in vivo. J. Urol. 1998, 159, 1390-1393. (b) Bush, P. A.; Aronson, W. J.; Buga, G. M.; Rajfer, J.; Ignarro, L. J. Nitric oxide is a potent relaxant of human and rabbit corpus cavernosum. J. Urol. 1992, 147, 1650-1655. (8) Bell, A. S.; Terrett, N. K. Pyrazolopyrimidinone antianginal agents. U.S. Patent 5426107. JM000081+ N-3-Substituted Imidazoquinazolinones Journal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1263 Copy with citationCopy as parenthetical citation