Ex Parte Ryde et al

Board of Patent Appeals and Interferences

Mar 10, 2011

10895405 (B.P.A.I. Mar. 10, 2011)

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