32-Ascomycinyloxyacetic acid derived immunosuppressants. Independence of immunophilin binding and immunosuppressive potency

Article abstract of DOI:10.1021/jm960066y

The potent immunosuppressant ascomycin (1b) was selectively alkylated at the C-32 carbinol, thus providing esters and amides of 32- ascomycinyloxyacetic acid (4, AOAA). These compounds present structural variation at the FKBP/calcineurin interface. While the native carboxylic acid 4 shows no activity in vitro, esters and simple amides of 4 exhibit potent immunosuppression in the human MLR assay. Moreover, amides show inhibitory activity in the rat popliteal lymph node hyperplasia assay. Surprisingly, FKBP binding was weakened by several orders of magnitude when secondary hydrophobic aryl amides of 4 were tested, while maintaining potent immunosuppressive efficacy in vitro.

Full text of DOI:10.1021/jm960066y

1764
J . Med. Chem. 1998, 41, 1764-1776
32-Ascom ycin yloxya cetic Acid Der ived Im m u n osu p p r essa n ts. In d ep en d en ce of
Im m u n op h ilin Bin d in g a n d Im m u n osu p p r essive P oten cy
Rolf Wagner,* Teresa A. Rhoades, Yat Sun Or, Benjamin C. Lane, Gin Hsieh, Karl W. Mollison, and J ay R. Luly
Abbott Laboratories, Pharmaceutical Products Division, Abbott Park, Illinois 60064
Received J anuary 22, 1996
The potent immunosuppressant ascomycin (1b) was selectively alkylated at the C-32 carbinol,
thus providing esters and amides of 32-ascomycinyloxyacetic acid (4, AOAA). These compounds
present structural variation at the FKBP/calcineurin interface. While the native carboxylic
acid 4 shows no activity in vitro, esters and simple amides of 4 exhibit potent immunosup-
pression in the human MLR assay. Moreover, amides show inhibitory activity in the rat
popliteal lymph node hyperplasia assay. Surprisingly, FKBP binding was weakened by several
orders of magnitude when secondary hydrophobic aryl amides of 4 were tested, while
maintaining potent immunosuppressive efficacy in vitro.
In tr od u ction
tein complexes. Structural similarities of FK506 and
RAP notwithstanding, the RAP/FKBP complex binds to
a unique effector protein,5 while the CsA/Cyp and
FK506/FKBP complexes converge upon a common tar-
get, calcineurin.6 The molecular and cellular mecha-
nisms of these agents have recently been reviewed.7 It
is now accepted that the cellular modes of action of these
three agents are defined by inhibition of the final protein
component.
Transplantation medicine has moved from a field of
speculation and obscurity several decades ago to a
discipline that now offers organ allografting as a readily
approved form of treatment. The acceptance of trans-
plantation as a viable treatment modality would have
been impossible without the development of immuno-
suppressive drugs. Due to their antiproliferative na-
ture, immunosuppressants have been haunted by nu-
merous toxic side effects,1 which include increased risk
of infection, tumor appearance and growth, and myelo-
suppression. Cyclosporin A (CsA), an immunosuppres-
sant approved for use in kidney transplatation in the
early 1980s, accelerated the acceptance of transplanta-
tion therapy due to a significantly improved safety pro-
file relative to previously known drugs such as steroids
and azathioprine. Nevertheless, despite being approved
for kidney transplantion, CsA showed kidney damaging
side effects, leaving much room for less toxic therapies.
Among the newer immunosuppressants, FK506 (1a ) has
recently been approved for use in liver transplantation,
and its structural relative rapamycin (2) is in clinical
trials. While these compounds provide alternatives to
cyclosporin therapy, they still demonstrate toxicity,
despite great initial hopes to the contrary.
Significant similarity in the toxicity/biological profile
of FK506 and CsA has emerged8 with reports implicat-
ing mechanism based toxicity.9 It is therefore likely
that calcineurin inhibition is the origin of toxicity. Cal-
cineurin is a ubiquitous protein with significant con-
centrations found in kidney and brain tissue. Among
the serious side effects being reported for FK506 and
CsA are nephrotoxicity and neurotoxicity.
We therefore defined the global objective of our pro-
gram as discovering an analogue of FK506 (1a ) or asco-
mycin (1b), which would retain potent immunosuppres-
sive activity, but at the same time exhibit reduced
systemic toxicity. Since 1a and 1b are potent immuno-
suppressants, we consequently realized the need to
identify a region of 1a or 1b which could tolerate alter-
ation without compromising immunosuppressive po-
tency. Also, since there was no structurally driven ra-
tionale to reduce the nephrotoxicity of 1a or 1b, the
chemistry would also need to provide access to diverse
functionality. Given that 1a (or 1b) functions as the
exquisite sandwiching partner between two proteins,
one would expect this exercise to be fraught with dif-
ficulty. It was therefore critical to identify the correct
region of the drug to be modified. Viewing rapamycin
as a distant analogue of FK506 inspired us to pursue
changes in the boundary surface of the FKBP/ascomycin
complex which was also positioned to interact with cal-
cineurin. This perspective justified the expectation that
such changes would alter the mode of action and/or the
toxicity of the ensuing analogues.10 Beneath the global
objective of discovering a less toxic immunosuppressant,
we then set out to identify compounds which altered the
binding characteristics of the target drug with FKBP
CsA, FK506 (1a ), and rapamycin (RAP, 2) exert their
immunosuppressive effects by inhibiting the prolifera-
tion of T lymphocytes, albeit by different mechanisms.
These effects are produced by the formation of three
unique trimolecular protein complexes, with the bio-
chemical fate determined by the last protein being
bound. FK506 and RAP bind to a peptidyl-prolyl-
isomerase (PPIase) FKBP-12,2 a necessary event for
inhibition of downstream T-cell proliferation, but which
by itself is not a sufficient condition.3 In a parallel
sense, CsA binds to cyclophilin (CyP), another PPIase,
an event that likewise is necessary but alone inadequate
for immunosuppression.4 It was later discovered that
all three agents, 1a , 2, and CsA, sandwich between
these PPIase’s and other protein partners. These drugs
apparently behaving as a molecular “glue” thus enable
the formation of the aforementioned trimolecular pro-
S0022-2623(96)00066-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/28/1998

Immunophilin Binding and Immunosuppressive Potency
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1765
and/or calcineurin. Herein we report modifications to
the 32-hydroxyl group of ascomycin (1b),11 which resides
at that interface.12 This moiety is perfectly positioned
between both proteins to allow modifications to be made
which could affect the stability of the consequent
trimolecular complex.
alkylated selectively. Novartis researchers report the
methylation of the C-32 alcohol by treating ascomycin
with diazomethane in the presence of Lewis acid.11a Of
particular interest is the work published by the Merck
group which utilized trichloroacetimidate esters to
prepare numerous C-32 ethers.11b,c On the basis of their
investigations, trichloroacetimidate esters of moieties
not capable of stabilizing a positive charge, such as a
glycolate, would not be expected to provide compounds
such as 3a or 3b (Scheme 1). We therefore turned our
attention to the use of alkyl diazoacetates, which had
been foreshadowed by the Novartis work. Ascomycin,
stirred in the presence of ethyl diazoacetate and Rh-
(OAc)2 in refluxing methylene chloride, provided 32-
ascomycinyloxyacetic acid ethyl ester 3a (AOAA ethyl
ester) in 50% yield. It has been shown that ascomycin
is unstable in the presence of strong bases, such as
sodium hydroxide, and therefore synthesis of AOAA (4)
via simple base hydrolysis was avoided.13 Moreover,
even treatment with mild acids can dehydrate the C-24
â-hydroxy ketone.14 Since hydrogenolysis of a benzyl
ester was anticipated to be uneventful, the synthesis of
AOAA benzyl ester (3b) was pursued. Alkylation of
ascomycin with benzyl diazoacetate at 0 °C gave 3b in
40% yield. Further analysis of the reaction mixture
showed that the bis-alkylated product 6 was formed. No
Ch em istr y
Since there was no clear structural rationale to solve
the toxicity problem associated with FK506 (1a ), reli-
ance upon a serendipitous discovery would be needed.
The incorporation of structural diversity at the C-32
position could probe effects on FKBP binding, immu-
nosuppression (in vitro and in vivo), and finally toxicity.
One intermediate that would efficiently provide syn-
thetic access to many different analogues is 32-ascomy-
cinyloxyacetic acid 4 (AOAA).
Ascomycin (1b) contains three hydroxyl groups, a
tertiary alcohol at C-10, and secondary alcohols at C-24
and C-32. Reports by other researchers have shown
that the C-32 carbinol of ascomycin or FK-506 can be
C-24-monoalkylated product was ever detected, dem-

1766 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Wagner et al.
Sch em e 1a
a
(a) Method A: RO2CCHN2, Rh(II)(OAc)2 (0.02 equiv), CH2Cl2; Method B: RO2CCH2I, Ag2O, CH3CN; (b) (R ) CH2Ph) H2 (1 atm), 10%
Pd(C) cat., EtOH; (c) Method A: EDAC, R1R2NH, CH2Cl2, 0 °C to room temperature; Method B: iBuOCOCl, N-methylmorpholine, R1R2NH,
THF, 0 °C to room temperature; (d) ArNH2 (12 equiv), EtMgBr (12 equiv), THF, -78 °C.
Sch em e 2a
a
(a) PhCH2O2CCHN2, Rh(II)(OAc)2 (cat.), CH2Cl2, room temperature; (b) HF (4 equiv), CH3CN, 0 °C.
onstrating that the 32-carbinol is more reactive than
the 24-hydroxyl group.
not add a measurable improvement, in practice the
chromatographic purification of final product was sim-
plified. Hydrogenolysis of benzyl ester 3b proceeded
cleanly, giving the pivotal carboxylic acid 4, in 70%
yield. Closer examination of this reaction mixture
revealed that the C-9 carbonyl was reduced to the
carbinol if the reaction was allowed to proceed too long
or at higher pressures.
Therefore, it was necessary to protect the C-24 as its
silyl ether 7, prior to alkylation (Scheme 2).15 Conse-
quently, the alkylation provided 8 in 70% yield. Re-
moval of the tert-butyldimethylsilyl group by brief
exposure to hydrogen fluoride at 0 °C provided 3b in
61% yield. While the overall yield of this procedure did

Immunophilin Binding and Immunosuppressive Potency
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1767
Ta ble 1.
rat PLN hyperplasia
IC50, nM (n)a
FKBP IC50
MLR IC50
/
inhibition ED50 mpk/d
compd
1a
X
FKBP binding
MLR inhibition
ip (n) (95% range)b
2.5 ( 0.2 (36)
2.1 ( 0.2 (32)
0.48 ( 0.09 (56)
5.2
8.4
0.1, 5
(0.06-0.16)
0.3, 10
(0.18-0.54)
2.1
1b
0.25 ( 0.05 (50)
2
1.1 ( 0.5 (6)
9.4 (1)
68. ( 28 (2)
4.0 ( 2.2 (2)
0.6 (1)
1.27 ( 0.30 (47)
0.91 ( 0.39 (6)
7.62 ( 4.26 (4)
140. ( 79.5 (3)
0.13 ( 0.10 (3)
0.9
10.
8.9
0.03
4.6
3a
3b
4
OC2H5
>3, 1
OCH2Ph
OH
>3, 1
>3, 1
9
NH2
0.3, 2
(0.20-0.50)
1.4, 1
1.9, 2
(1.2-3.2)
1.2, 2
(0.71-3.11)
>3, 1
>3, 1
>3, 3
10
11
NHPh
170 ( 110 (7)
2.9 ( 1.5 (3)
0.29 ( 0.16 (9)
0.26 ( 0.07 (4)
590.
11.
N(CH3)Ph
12
NHCH2Ph
48. ( 23. (4)
0.79 ( 0.26 (7)
61.
13
14
15
N(CH3)CH2Ph
NH(CH2)2Ph
N(CH3)(CH2)2Ph
5.6 ( 2.3 (4)
13. ( 0.34 (2)
0.4 ( 0.1 (2)
0.09 ( 0.04 (4)
0.34 ( 0.21 (6)
0.64 ( 0.17 (5)
62.
38.
0.63
a
b
Data reported as the mean ( SD for n determinations as noted. 95% confidence limits for n determinations.
Condensations with amines to provide amides 5 were
run uneventfully using either EDAC or isobutyl chlo-
roformate as the activating agent. One exception to this
sequence resulted from the direct addition of the mag-
nesium bromide salt of 3,4-dichloroaniline to the ester
3b at low temperature.16 Treatment of esters with alu-
minum amides generated by the pretreatment of anilines
with trimethylaluminum gave reaction mixtures con-
taining more products than the ammonolyses mediated
by the magnesium salts.17 While more direct, this pro-
cedure was explored very late in the development of the
aryl amide series and therefore remained less favored
than conventional amine acylation through AOAA (4).
Upon scaleup, the impractical nature of the current
strategy becomes apparent. In addition to the large
glassware and unwieldy solvent requirements, the large
volume of the benzyl diazoacetate solution needs to be
added over 14 h with a syringe pump. Furthermore,
the introduction of protection/deprotection steps re-
sulted in a process that was deemed too inefficient for
the production of 100 g quantities of 3b. A more
practical synthesis of benzyl ester 3b, without the need
for protecting the C-24 carbinol, resulted from Ag2O
promoted alkylation of ascomycin using benzyl iodoac-
etate (Scheme 1, method B of step a). This reaction was
routinely run on 100-200 g of ascomycin (1b).
of 1a and 1b, it is less potent in the in vivo model of
lymphoproliferation (PLN).
A crude comparison index can be constructed that
illustrates the influence of FKBP binding on immuno-
suppressive potency. Thus, the ratio of the FKBP IC50
to HuMLR IC50 is 5.2 and 8.4 for FK506 (1a ) and
ascomycin (1b), respectively. Therefore, a new com-
pound displaying a ratio between 5.2 and 8.4 can be said
to display consistent behavior with respect to 1a or 1b.
If one compares the FKBP/HuMLR ratios for 1a (or 1b)
and 2, a significant difference between FK506 and
rapamycin is illustrated, a noteworthy result since
divergent mechanisms of immunosuppression have been
demonstrated. Esters 3a and 3b bind less avidly to
FKBP than the preceding compounds; however the
proportional decrease of the in vitro immunosuppressive
potency (HuMLR) results in FKBP/HuMLR ratios which
fall near the expected range.
The first compound displaying aberrant behavior is
carboxylic acid 4, with a ratio of 0.03. Although FKBP
affinity is reduced only 2-fold, immunosuppressive
potency (HuMLR IC50) is 2-3 orders of magnitude
weaker than 1a or 1b. In vivo (PLN assay), 4 gave no
measurable immunosuppression at 3 mpk/day. While
it is not clear that the inactivity of 4 could be attributed
to its inability to penetrate cell membranes or tissue
barriers, esters of AOAA (4) should transverse these
restrictions. Nevertheless, esters 3a and 3b, which
could conceivably be metabolized to 4, did not have
measurable in vivo activity at the same dose either.
Changing the functional group at “X” (Table 1) to an
amide alters the in vivo response significantly for
compounds 9, 10, 11, and 12, while 13, 14, and 15
remain inactive at 3 mpk/day in the PLN assay. All of
the amides in Table 1 have subnanomolar MLR IC50’s
ranging from 0.09 to 0.79 nM. Furthermore, each amide
except for 10, 12, and perhaps 14 have unremarkable
FKBP IC50’s. These three exceptional amides are the
Resu lts a n d Discu ssion
The assays used to evaluate the biological conse-
quences of these structural modifications, the human
mixed lymphocyte response (HuMLR), human FKBP
binding inhibition, and Brown Norway to Lewis rat
popliteal lymph node hyperplasia inhibition assay (PLN),
have been described previously.18 Table 1 illustrates
that FK506 (1a ) and ascomycin (1b) are comparable in
vitro and in vivo. Although rapamycin (2) displays
similar FKBP binding and comparable in vitro immu-
nosuppressive potency (HuMLR) with respect to those

1768 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Wagner et al.
Ta ble 2.
IC50, nM, (n)a
FKBP binding
compd
Y
MLR inhibition
FKBP IC50/MLR IC50
8.4
590.
4.3
48.
1b
10
16
17
18
19
20
21
22
23
24
25
26
27
28
29
2.1 ( 0.2 (32)
170 ( 110 (7)
2.0 (1)
0.25 ( 0.05 (50)
0.29 ( 0.16 (9)
0.47 ( 0.15 (4)
0.05 ( 0.01 (4)
0.10 ( 0.04 (6)
0.16 ( 0.05 (4)
0.14 ( 0.04 (11)
0.58 ( 0.19 (10)
0.55 ( 0.36 (4)
0.07 ( 0.03 (4)
0.04 ( 0.02 (4)
0.14 ( 0.05 (4)
0.23 ( 0.10 (4)
0.16 ( 0.09 (4)
0.03 ( 0.01 (6)
0.02 ( .003 (3)
Ph
cyclohexyl
2-pyridinyl
3-pyridinyl
4-pyridinyl
p-(HO)Ph
p-(4-morpholino)Ph
3,4-dichloro-Ph
p-(Cl)Ph
2.4 (1)
3.3 ( 2.1 (3)
3.2 ( 1.8 (3)
16. ( 5.5 (5)
19. ( 3.1 (3)
500. ( 80. (3)
180. ( 45. (2)
50. ( 13. (2)
470. (1)
33.
20.
110.
33.
910.
1400.
1300.
3400.
2700.
2500.
1500.
3600.
p-(CH3O)Ph
m-(CF3)Ph
p-(CF3)Ph
570. (1)
180. (1)
m-(F)Ph
p-(F)Ph
46. ( 0.80 (2)
71. ( 3.4 (2)
p-(CH3)Ph
a
Data reported as the mean ( SD for n determinations as noted.
first known ascomycin analogues shown to retain sub-
nanomolar immunosuppressive efficacy, while showing
marked loss of affinity for the immunophilin. Indeed,
the FKBP/HuMLR ratios for 10, 12, and 14 are 590, 61,
and 38, respectively. These amides likely adopt a
predominantly E-amide geometry, which must disrupt
binding to FKBP. This effect is amplified as the
hydrophobic aryl ring gets closer to the amide nitrogen,
with the greatest binding disruption occurring with
phenyl amide 10.19 The option of a Z-amide rotamer,
provided by tertiary amides 11, 13, and 15, results in
profound increases in FKBP affinity.
boost in immunosuppressive potency is obtained, with
the FKBP/HuMLR ratios being 110 and 33, respectively.
As hydrophobic substituents are added (22-29), this
immunosuppressive boost is retained in most cases,
while FKBP binding is further disrupted, in some cases
to nearly micromolar levels. Consequently, these two
effects elevate the FKBP/HuMLR ratios from 910 to
3600. Clearly, significant disruption of the FKBP/drug/
calcineurin mating surfaces can happen without com-
promising MLR potency.
Con clu sion
Disparate behavior, such as has been discovered for
the phenyl amide 10, could enable the discovery of an
agent with an altered mechanism of action and safety
profile. Toward that end, additional aryl amides were
synthesized, and the impact on FKBP binding and
immunosuppressive efficacy was determined (Table 2).
Without exception, each analogue exhibited a HuMLR
IC50 at a subnanomolar concentration ranging from 0.02
to 0.58 nM. On the other hand, FKBP binding ranged
from 2.0 to 570 nM, and the FKBP/HuMLR ratios
ranged from 4.3 to 3600, demonstrating a profound lack
of correlation between FKBP binding and in vitro
immunosuppressive efficacy.
The amide must satisfy several criteria if dissociation
from FKBP is to be observed. These are secondary
amide structure and hydrophobic substitution. While
no protracted effort has been made to show the require-
ment for aromaticity, the FKBP affinity of the cyclohexyl
amide 16 resembles the FKBP affinity of ascomycin (1b)
much more than that of the phenyl amide 11. It is
therefore likely that aromaticity is a third requirement
for attenuated immunophilin binding.
Is it possible to create a potent immunosuppressant
based on the ascomycin framework that is profoundly
less nephrotoxic than FK506 (1a )? This remains the
critical question that compels researchers in this area
to continue searching for such an agent. The fact that
rapamycin (2) is immunosuppressive, binds FKBP, and
yet operates via a novel mechanism suggests that great
opportunity awaits the medicinal chemist who is willing
to alter the mating surface of the FKBP-drug complex.
Indeed, in recent reports Merck researchers have un-
veiled 32-O-(1-hydroxyethylindol-5-yl)ascomycin, a com-
pound that shows approximately a 10-fold decrease in
FKBP affinity, while maintaining equivalent in vitro
and in vivo potency when compared with FK506 (1a ).
Most interestingly, an improved toxicity profile appears
to be emerging from this agent.20 With the AOAA-
derived amides, we have also achieved significant bind-
ing disruption at the immunophilin site, while demon-
strating surprising immunosuppressive activity in human
T-lymphocytes. The impact of these manipulations and
others on kidney function will be the subject of future
reports.
Pyridyl amides 17, 18, and 19 are unremarkable,
indicating that aryl rings with hydrogen bonding capa-
bilities display FKBP affinities similar to that of asco-
mycin. Furthermore, AOAA phenolic amide 20 and
AOAA p-(4-morpholino)-phenyl amide 21 show affinities
to FKBP that are less than 10-fold weaker than asco-
mycin (1b). In both of these cases, however, a slight
Exp er im en ta l Section
Gen er a l Meth od s. Unless otherwise specified, proton
magnetic resonance spectra were run as chloroform-d solutions
at 500 MHz. Carbon-13 nuclear magnetic resonance spectra
were run as chloroform-d solutions at 125 MHz. Chloroform-d
was used as an internal standard. DEPT (distortionless

Immunophilin Binding and Immunosuppressive Potency
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1769
enhancement by polarization transfer) was employed to assign
carbon multiplicities in some cases. Melting points are uncor-
rected. Mass spectra, infrared spectra, and the above deter-
minations were performed by the Analytical Research Depart-
ment, Abbott Laboratories. Elemental analyses were performed
either by the Analytical Research Department, Abbott Labo-
ratories, or Robertson Microlit Laboratories, Inc., Madison, NJ .
mL) and brine (400 mL). The aqueous portions were extracted
with additional ethyl acetate (400 mL), and the combined
organics were dried (Na2SO4) and concentrated in vacuo to a
brown oil. This was passed through a pad of basic alumina
(100 mL) and eluted with hexanes (200 mL). Concentration
of the eluate provided benzyl iodoacetate as an amber oil (171
g) which was sufficiently pure to use in the next step.
Analytical thin-layer chromatography was done on 2 × 6
cm Kieselgel 60 F-254 plates precoated with 0.25 mm thick
silica gel distributed by E. Merck. Visualization was ac-
complished with a solution consisting of ammonium molybdate
(50 g) and ceric sulfate (20 g) in 10% sulfuric acid (2 L) followed
by charring on a hot plate, or with short-wavelength ultraviolet
light. Unless otherwise specified, column chromatography was
performed on silica gel (Kieselgel 60, 70-230 mesh) from E.
Merck. Preparative HPLC purification was performed at
ambient temperature on a 20 × 300 mm column packed with
YMC 15 µm 60-Å spherical silica gel, eluting at 10-20 mL/
min, using a differential refractometer to monitor eluate
composition. The term in vacuo refers to solvent removal via
a rotary evaporator at 30 mmHg. All final products were
lyophylized from benzene. Analytical samples were further
dried by final evacuation at 0.1 mmHg for 18 h.
(b) In a three-necked 2 L round-bottom flask equipped with
an overhead stirrer, ascomycin 1b (100 g, 0.13 mol, crystalline
material completely dissolved in methylene chloride then
concentrated to a dry foam) and benzyl iodoacetate (171 g, 0.62
mol, 4.9 equiv) were mixed together and then dissolved
completely in acetonitrile (50 mL).21 The thick solution was
cooled to 0 °C whereupon Ag2O (119 g, 0.51 mol, 4 equiv) was
added portionwise over 15 min (ca. 15 additions).22 After the
addition was complete and the reaction was mixed (5 min after
last addition), the ice bath was removed and the reaction
allowed to stir at ambient temperature for 7 days. Diethyl
ether (400 mL) was added to the reaction mixture, and this
was then poured over silica gel (600 mL), mixed and allowed
to air-dry overnight. A 3 L coarse fritted Buchner funnel was
charged with silica gel (2 L) and the adsorbed silica carefully
layered over the dry fresh bed, followed by a filter paper disk.
The silica pad was eluted with CH2Cl2 (3 L), CH2Cl2:CH3CN
(9:1, 4 L), CH2Cl2:CH3CN (6:1, 4 L), CH2Cl2:CH3CN (3:1, 8 L),
and acetone (4 L), collecting 1 L fractions throughout. Eluate-
containing product was pooled and concentrated to semipure
3b (72 g), while ascomycin (27 g) was isolated from the acetone
eluate. Final purification was achieved by another passage
through dry-packed silica gel (2 L) in a Buchner funnel.
Elution with CH2Cl2 (2 L), hexane:acetone (4:1, 6 L), and
hexane:acetone (3:1, 8 L), collecting 1 L fractions throughout,
resulted in pure solutions of 3b which were combined and
concentrated to a pale yellow foam (61 g, 65 mmol): IR (CDCl3)
3510, 2930, 1740, 1695, 1642, 1450 cm-1; 1H NMR (500 MHz)
CDCl3 δ (rotamers 2:1) 7.39-7.29 (comp, 5H), 5.34 (d, 0.67H,
J ) 3.0 Hz), 5.22 (m, 0.33H), 5.21 (d, 1H, J ) 12.0 Hz), 5.18
(d, 1H, J ) 12.0 Hz), 5.11-4.98 (mult, 2H), 4.77 (d, 0.67H, J
) 2.5 Hz), 4.72 (s, 0.33H), 4.62 (d, 1H, J ) 6.0 Hz), 4.45 (br d,
0.67H, J ) 15.0 Hz), 4.39 (d, 1H, J ) 16.0 Hz), 4.34 (d, 1H, J
) 16.0 Hz), 4.25 (d, 1H, J ) 2.5 Hz), 3.98-3.87 (mult, 1.33H),
3.74 (br d, 0.33H, J ) 14.5 Hz), 3.69 (d, 0.67H, J ) 10.0 Hz),
3.60 (mult, 1H), 3.51-3.14 (comp, 4.33H), 3.42 (s, 2H), 3.41
(s, 3H), 3.40 (s, 1H), 3.35 (s, 1H), 3.31 (s, 2H), 3.04 (comp,
0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.75 (dd, 0.33H, J
) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s, 1H), 1.66 (s,
1H), 1.65 (s, 2H), 1.61 (s, 2H), 1.01 (d, 2H, J ) 6.0 Hz), 0.98
(d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz), 0.92 (d, 1H, J )
7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd, 3H, J ) 7.5, 7.5
Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125 MHz) δ 9.4, 11.7,
14.1, 15.8, 16.2, 20.4, 21.1, 24.1, 24.5, 26.3, 27.6, 30.3, 30.8,
32.7, 32.8, 34.4, 34.5, 36.3, 39.2, 39.6, 43.1, 48.6, 53.4, 54.6,
56.3, 56.6, 57.1, 66.3, 68.5, 70.1, 72.8, 73.6, 75.1, 76.8, 82.7,
83.6, 96.9, 123.0, 128.3, 128.4, 128.5, 129.5, 132.3, 135.6, 138.7,
164.7, 168.9, 171.0, 196.2, 213.4; MS (FAB) m/z M + H - H2O
) 922, M + K ) 978. Anal. Calcd for C52H77NO14: C, 66.43;
H, 8.26; N, 1.49. Found: C, 66.12; H, 8.14; N, 1.41.
With the exception of amines, solvents and reagents were
purchased from Aldrich Chemical Co. and were used without
further purification unless otherwise specified. All amines
were dried over molecular sieves (4 Å) for at least 24 h prior
to use. Tetrahydrofuran (THF) was distilled from sodium
benzophenone ketyl.
32-Ascom ycin yloxya cetic Acid Eth yl Ester (3a ).
A
solution of ascomycin (0.5 g, 0.6 mmol) in dichloromethane (10
mL) containing rhodium(II) acetate dimer (3.0 mg, 0.01 mmol,
0.02 equiv) was refluxed while ethyl diazoacetate (66 uL, 0.6
mmol) in dichloromethane (1 mL) was added dropwise. After
the addition was complete, the reaction was refluxed for 30
min and additional ethyl diazoacetate (132 uL, 1.26 mmol) in
dichloromethane (1.5 mL) was added dropwise with reflux
continuing 30 min after complete addition. Solvent was
removed in vacuo and the residue purified by HPLC on silica
gel eluting with hexane:acetone (3:1). Fractions containing
the desired product were pooled, concentrated, dissolved in
CCl4, and concentrated to constant weight under high vacuum
to give the desired product (274 mg) as an oil in 50% yield:
IR (CDCl3) 3500, 2930, 1742, 1700, 1645, 1452 cm-1; 1H NMR
(500 MHz) CDCl3 δ (rotamers 2:1) 5.34 (d, 0.67H, J ) 3.0 Hz),
5.21 (d, 0.33H, J ) 3.0 Hz), 5.09-4.98 (mult, 2H), 4.82 (d, 1H,
J ) 2.5 Hz), 4.61 (d, 1H, J ) 6.0 Hz), 4.44 (br d, 0.67H, J )
15.0 Hz), 4.33 (d, 1H, J ) 16.0 Hz), 4.29 (d, 1H, J ) 16.0 Hz),
4.25 (s, 1H), 4.23 (d, 1H, J ) 7.0 Hz), 4.20 (d, 1H, J ) 7.0 Hz),
3.98-3.87 (mult, 1.33H), 3.74 (br d, 0.33H, J ) 14.5 Hz), 3.68
(d, 0.67H, J ) 10.0 Hz), 3.58 (mult, 1H), 3.50-3.06 (comp,
4.33H), 3.42 (s, 2H), 3.41 (s, 1H), 3.40 (s, 2H), 3.39 (s, 1H),
3.35 (s, 1H), 3.31 (s, 2H), 3.03 (comp, 0.67H), 2.79 (dd, 0.67H,
J ) 17.5, 4.5 Hz), 2.74 (dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-
0.74 (comp, 26H), 1.67 (s, 1H), 1.64 (s, 3H), 1.61 (s, 2H), 1.29
(dd, 3H, J ) 7.5, 7.5 Hz), 1.01 (d, 2H, J ) 6.0 Hz), 0.98 (d, 1H,
J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz), 0.92 (d, 1H, J ) 7.5 Hz),
0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd, 3H, J ) 7.5, 7.5 Hz), 0.85
(d, 1H, J ) 7.5 Hz); 13C NMR (125 MHz) δ 9.4, 11.7, 14.1, 14.2,
15.8, 16.2, 20.5, 21.1, 24.2, 24.6, 26.3, 27.6, 30.3, 30.8, 32.7,
32.9, 33.6, 34.6, 36.4, 39.2, 39.7, 43.1, 48.7, 54.7, 56.3, 56.6,
56.9, 57.2, 60.6, 68.5, 70.1, 72.9, 73.7, 75.2, 77.2, 82.8, 83.6,
97.0, 123.1, 129.6, 132.4, 138.7, 164.7, 169.0, 171.1, 196.1,
213.5; MS (FAB) m/z M + K ) 916. Anal. Calcd for
32-Ascom ycin yloxya cetic Acid (4). A Parr shaker flask
was charged with AOAA benzyl ester 3b (41 g, 44 mmol), 10%
Pd/C (4.7 g), and ethanol (1.1 L) and then flushed with nitrogen
for 10 min. The reaction vessel was shaken under a hydrogen
atmosphere (4 atm) for 15 min. The mixture was filtered, and
the catalyst was extracted once with additional ethanol (1 L),
whereupon the filtrates were combined and the solvent was
removed in vacuo (41 g). A column was packed with dry silica
gel (5.2 L, E. Merck Kieselgel 60, 230-400 mesh) and prewet-
ted with CH2Cl2:2-propanol (19:1) containing 0.5% CH3CO2H.
Elution with CH2Cl2:2-propanol (19:1, 12 L) containing 0.5%
CH3CO2H, followed by CH2Cl2:2-propanol (9:1, 12 L) containing
0.5% CH3CO2H, collecting 1 L fractions throughout, provided
recovered 3b (1.7 g) and AOAA 4 (33 g, 39 mmol) in 88%
yield: IR (CDCl3) 3450, 1740 cm-1; 1H NMR (500 MHz) CDCl3
δ (rotamers 2:1) 5.43 (d, 0.67H, J ) 3 Hz), 5.22 (d, 0.33H, J )
3 Hz), 5.03 (mult, 2H), 4.88 (br s, 1H), 4.63 (d, 1H, J ) 5.0
C
47H75NO14‚1.0CCl4: C, 54.70; H, 7.33; N, 1.36. Found: C,
54.42; H, 7.22; N, 1.26.
32-Ascom ycin yloxya cetic Acid Ben zyl Ester (3b). (a)
A mixture of sodium iodide (608 g, 4.0 mol) in acetone (1.6 L)
was heated to reflux for 5 min and cooled to room temperature.
Benzyl bromoacetate (114 mL, 0.72 mol) was added, and the
reaction was stirred for 30 min. The solvent was removed in
vacuo, and the resulting slurry was partitioned between water
(800 mL) and ethyl acetate (400 mL). The organic layer was
washed sequentially with saturated sodium bisulfite (2 × 400

1770 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Wagner et al.
Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.30 (d, 1H, J ) 17.5 Hz),
4.26 (s, 1H), 3.99 (d, 0.67H, J ) 17.5 Hz), 3.98 (d, 0.33H, J )
17.5 Hz), 3.95-3.82 (mult, 1.33H), 3.75 (br d, 0.33H, J ) 15.0
Hz), 3.68 (d, 0.67H, J ) 10 Hz), 3.59 (mult, 1H), 3.55-2.95
(mult, 5H), 3.50 (s, 3H), 3.40 (s, 2H), 3.39 (s, 1H), 3.35 (s, 1H),
3.31 (s, 2H), 2.79 (dd, 0.67H, J ) 15.0, 3.0 Hz), 2.73 (dd, 0.33H,
J ) 15.0, 3.0 Hz), 2.37-0.83 (comp, 26H), 1.69 (s, 1H), 1.66 (s,
2H), 1.64 (s, 1H), 1.62 (s, 2H), 1.01 (d, 2H, J ) 7.5 Hz), 0.98
(d, 1H, J ) 7.5 Hz), 0.95 (d, 2H, J ) 7.0 Hz), 0.93 (d, 1H, J )
7.0 Hz), 0.89 (d, 2H, J ) 7.0 Hz), 0.88 (dd, 3H, J ) 7.5, 7.5
Hz), 0.85 (d, 1H, J ) 7.0 Hz); 13C NMR (125 MHz) δ (rotamers)
213.5, 213.4, 196.1, 192.5, 172.1, 169.0, 168.7, 165.8, 164.7,
139.7, 138.8, 133.1, 132.5, 128.7, 127.1, 123.8, 123.0, 98.7, 97.1,
83.5, 82.0, 77.8, 77.2, 76.6, 75.2, 73.7, 73.6, 72.9, 72.2, 70.0,
68.9, 67.6, 57.6, 57.0, 56.6, 56.5, 56.3, 56.0, 55.0, 54.7, 52.7,
48.7, 48.5, 43.9, 43.4, 43.1, 40.3, 39.7, 39.2, 35.5, 35.2, 34.7,
34.3, 33.7, 33.0, 32.7, 32.5, 30.4, 29.5, 27.5, 26.3, 26.2, 26.0,
24.6, 24.5, 24.2, 21.1, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8, 15.6,
14.2, 14.1, 11.7, 9.8, 9.5; MS (FAB) m/z M + K ) 888. Anal.
10 (0.80 g, 73% yield): IR (KBr) ν 3340, 2940, 2880, 2830, 1740,
1
1690, 1645 cm-1; H NMR (500 MHz) CDCl3 δ (rotamers 2:1)
9.59 (s, 0.33H), 9.57 (s, 0.67H), 7.62 (d, 2H, J ) 8.0 Hz), 7.34
(dd, 2H, J ) 8.0, 8.0 Hz), 7.12 (dd, 2H, J ) 8.0, 8.0 Hz), 5.34
(d, 0.67H, J ) 3.0 Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97
(mult, 2H), 4.88 (d, 1H, J ) 2.5 Hz), 4.62 (d, 1H, J ) 6.0 Hz),
4.45 (br d, 0.67H, J ) 15.0 Hz), 4.25 (d, 0.67H, J ) 16.5 Hz),
4.24 (d, 0.33H, J ) 16.0 Hz), 4.24 (s, 1H), 4.02 (d, 0.67H, J )
17.0 Hz), 4.01 (d, 0.33H, J ) 17.5 Hz), 3.98-3.87 (mult, 1.33H),
3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H, J ) 10.0 Hz),
3.59 (mult, 1H), 3.50-3.12 (comp, 4.33H), 3.57 (s, 3H), 3.39
(s, 2H), 3.38 (s, 1H), 3.35 (s, 1H), 3.31 (s, 2H), 3.03 (comp,
0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74 (dd, 0.33H, J
) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s, 1H), 1.65 (s,
2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J ) 6.0 Hz), 0.98
(d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz), 0.92 (d, 1H, J )
7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd, 3H, J ) 7.5, 7.5
Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125 MHz) δ (rotamers)
213.2, 213.1, 196.1, 192.6, 169.0, 168.9, 168.6, 165.7, 164.6,
139.5, 138.6, 137.8, 132.7, 132.2, 129.1, 129.0, 128.8, 124.0,
123.2, 123.0, 119.6, 98.5, 96.9, 82.1, 82.0, 77.7, 77.2, 76.5, 75.1,
73.5, 72.8, 72.2, 69.9, 68.9, 67.8, 57.4, 56.8, 56.7, 56.5, 56.2,
55.9, 54.8, 54.6, 52.6, 48.6, 48.3, 43.8, 43.4, 43.2, 40.1, 39.7,
39.1, 35.5, 35.5, 35.3, 34.4, 34.4, 33.6, 32.9, 32.6, 32.4, 31.4,
30.3, 28.9, 27.5, 26.2, 26.1, 25.9, 24.4, 24.4, 24.1, 22.5, 21.0,
20.7, 20.3, 19.4, 16.1, 15.9, 15.7, 15.6, 14.1, 14.0, 11.6, 9.7, 9.3;
mass spectrum (FAB), 963 [M + K]+. Anal. Calcd for
C51H76N2O13: C, 66.21; H, 8.27; N, 3.02. Found: C, 66.99; H,
8.94; N, 2.81.
Calcd for
C45H71NO14‚H2O: C, 62.61; H, 8.80; N, 1.33.
Found: C, 62.26; H, 8.47; N, 1.61.
32-Ascom ycin yloxya ceta m id e (9). To a stirred solution
of AOAA (4) (1.0 g, 1.2 mmol) in dry THF (4 mL) at 0 °C was
added N-methylmorpholine (130 µL, 1.2 mmol) followed by
isobutyl chloroformate (150 µL, 1.2 mmol). After 30 min,
concentrated NH4OH (160 µL, 2.4 mmol) was added, and the
reaction was warmed gradually to room temperature, followed
by stirring overnight. The mixture was partitioned between
EtOAc (150 mL) and 1 N HCl (150 mL). The organic layer
was washed with an additional portion of 1 N HCl (150 mL),
saturated NaHCO3 solution (2 × 150 mL), and then brine (150
mL). The organic layer was dried (Na2SO4), filtered, and
concentrated in vacuo. The crude material was dissolved in
CH2Cl2 and then passed through a pad of silica gel (50 g)
eluting with CH2Cl2 (300 mL), 2:1 hexane:acetone (600 mL),
1:1 hexane:acetone (300 mL), and then finally 1:2 hexane:
acetone (600 mL), collecting 20 mL fractions throughout.
Eluate containing the desired product was pooled and concen-
trated in vacuo. Further purification by HPLC eluting with
1:2 hexane:acetone afforded pure 9 (0.61 g, 61% yield): IR
(KBr) ν 3425, 2960 (sh), 2920, 2850 (sh), 2820 (sh), 1740, 1670
N-Met h yla n ilin e-(32-Ascom ycin yloxya cet ic
a cid )
a m id e (11). N-Methylaniline was coupled to AOAA (4) on a
0.94 mmol scale according to the method used for the synthesis
of 9. The silica pad was eluted with CH2Cl2 (100 mL) followed
by 1:1 hexane:acetone (150 mL). Fractions containing the
desired product were pooled and concentrated in vacuo to give
640 mg of yellow foam which was further purified by HPLC
eluting with 3:2 hexane:acetone to provide 11 (0.48 g, 54%
yield): IR (KBr) ν 3425, 2960 (sh), 2930, 2850, 2820, 1740,
1700, 1643, 1598 cm-1; 1H NMR (500 MHz) CDCl3 δ (rotamers
2:1) 7.42 (m, 2H), 7.35 (m, 1H), 7.21 (m, 2H), 5.23 (d, 0.67H,
J ) 3.0 Hz), 5.19 (d, 0.33H, J ) 3.0 Hz), 5.07-4.97 (mult, 2H),
4.82 (d, 1H, J ) 2.5 Hz), 4.60 (d, 1H, J ) 6.0 Hz), 4.43 (br d,
0.67H, J ) 15.0 Hz), 4.23 (s, 1H), 4.09 (br s, 2H), 3.98-3.87
(mult, 1.33H), 3.73 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H,
J ) 10.0 Hz), 3.59 (mult, 1H), 3.50-3.09 (comp, 4.33H), 3.40
(s, 2H), 3.39 (s, 1H), 3.36 (s, 1H), 3.35 (s, 2H), 3.30 (s, 2H),
3.28 (s, 1H), 3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 15.5, 7.5
Hz), 2.74 (dd, 0.33H, J ) 15.5, 7.5 Hz), 2.63 (s, 0.25H), 2.37-
0.74 (comp, 26H), 2.19 (s, 0.25H), 2.18 (s, 2.5H), 1.65 (s, 1H),
1.64 (s, 1H), 1.62 (s, 2H), 1.60 (s, 2H), 1.01 (d, 2H, J ) 7.0
Hz), 0.97 (d, 1H, J ) 7.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz), 0.91 (d,
1H, J ) 7.5 Hz), 0.87 (dd, 3H, J ) 7.5, 7.5 Hz), 0.86 (d, 2H, J
) 6.5 Hz), 0.84 (d, 1H, J ) 6.5 Hz); 13C NMR (125 MHz) δ
(rotamers) 213.5, 213.4, 196.1, 192.6, 169.8, 168.9, 168.6, 165.8,
164.7, 142.9, 139.6, 138.7, 132.2, 131.7, 129.7, 129.6, 129.7,
127.9, 127.1, 123.3, 123.1, 98.6, 97.0, 83.6, 83.6, 82.4, 77.8, 77.2,
76.7, 75.2, 73.7, 73.7, 72.9, 72.3, 70.1, 69.2, 69.1, 57.5, 57.3,
57.2, 56.9, 56.6, 56.3, 56.1, 55.0, 54.7, 52.7, 48.7, 48.5, 43.8,
43.5, 43.1, 40.3, 39.7, 39.2, 37.3, 36.5, 36.4, 35.5, 34.6, 33.6,
32.9, 32.8, 32.6, 30.8, 30.2, 30.2, 29.3, 27.6, 26.4, 26.2, 26.0,
24.6, 24.5, 24.2, 21.1, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8, 15.7,
14.2, 14.1, 11.7, 9.8, 9.4; mass spectrum (FAB), 977 [M + K]+.
1
(sh), 1643 cm-1; H NMR (500 MHz) CDCl3 δ (rotamers 2:1)
7.80 (m, 1H), 5.44 (br s, 1H), 5.34 (d, 0.67H, J ) 3.0 Hz), 5.22
(d, 0.33H, J ) 3.0 Hz), 5.09-4.97 (mult, 2H), 4.62 (d, 1H, J )
6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.25 (br s, 1H), 4.13
(d, 0.67H, J ) 16.5 Hz), 4.12 (d, 0.33H, J ) 16.0 Hz), 3.96 (d,
0.67H, J ) 17.0 Hz), 3.95 (d, 0.33H, J ) 17.5 Hz), 3.98-3.87
(mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H,
J ) 10.0 Hz), 3.59 (mult, 1H), 3.50-3.12 (comp, 4.33H), 3.45
(s, 3H), 3.44 (s, 2H), 3.39 (s, 1H), 3.35 (s, 1H), 3.31 (s, 2H),
3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74
(dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s,
1H), 1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J )
6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz),
0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd,
3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125
MHz) δ (rotamers) 213.4, 213.4, 196.1, 192.6, 174.0, 169.0,
168.7, 165.8, 164.7, 139.6, 138.7, 132.8, 132.2, 129.2, 129.2,
123.3, 123.1, 98.7, 97.1, 82.3, 82.2, 77.9, 77.2, 76.6, 75.2, 73.7,
73.6, 72.9, 72.3, 70.0, 69.0, 68.0, 57.5, 57.0, 56.8, 56.6, 56.3,
56.0, 55.0, 54.7, 52.7, 48.7, 48.5, 43.9, 43.5, 43.3, 40.4, 39.8,
39.2, 35.8, 35.7, 35.5, 34.6, 34.5, 33.7, 33.0, 32.7, 32.6, 30.5,
29.3, 27.5, 26.3, 26.2, 26.0, 24.6, 24.5, 24.2, 21.1, 20.8, 20.4,
19.5, 16.2, 16.0, 15.7, 15.8, 14.2, 14.1, 11.7, 9.8, 9.5; mass
Anal. Calcd for C52H78N3O13
: C, 66.50; H, 8.37; N, 2.98.
Found: C, 66.31; H, 8.50; N, 2.94.
spectrum (FAB), 887 [M + K]+. Anal. Calcd for C45H72N2O13
:
Ben zyla m in e-(32-Ascom ycin yloxya cetic a cid )a m id e
(12). Benzylamine was coupled to AOAA (4) on a 1.2 mmol
scale according to the method used for the synthesis of 9. The
silica pad was eluted with CH2Cl2 (100 mL) followed by 2:1
hexane:acetone (300 mL). Fractions containing the desired
product were pooled and concentrated in vacuo to give 1.1 g
of yellow foam which was further purified by HPLC eluting
with 3:2 hexane:acetone to give semipure 12 (0.8 g). A final
purification by HPLC eluting with 4% IPA/CH2Cl2 afforded
pure 12 (0.45 g, 41% yield): IR (KBr) ν 3420, 2950, 2920, 2865,
C, 63.65; H, 8.54; N, 3.29. Found: C, 63.95; H, 8.71; N, 3.15.
An ilin e-(32-Ascom ycin yloxya cetic a cid )a m id e (10).
Aniline was coupled to AOAA (4) on a 1.2 mmol scale according
to the method used for synthesis of 9. The silica pad was
eluted with CH2Cl2 (150 mL) followed by 3:1 hexane:acetone
(100 mL) and then 2:1 hexane:acetone (200 mL). Fractions
containing the desired product were pooled and concentrated
in vacuo to give 1.03 g of yellow foam which was further
purified by HPLC eluting with 5:2 hexane:acetone to provide

Immunophilin Binding and Immunosuppressive Potency
2820, 1740, 1700, 1645 cm-1 1H NMR (500 MHz) CDCl3
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1771
;
δ
(br s, 1H), 4.62 (d, 1H, J ) 6.5 Hz), 4.45 (br d, 0.67H, J ) 14.0
Hz), 4.26 (s, 1H), 4.11 (d, 1H, J ) 17.5 Hz), 4.0-3.86 (mult,
1.33H), 3.93 (d, 0.67H, J ) 17.5 Hz), 3.92 (d, 0.33H, J ) 17.5
Hz), 3.75 (br d, 0.33H, J ) 14.0 Hz), 3.68 (d, 0.67H, J ) 10
Hz), 3.67-3.56 (mult, 2H), 3.56-3.42 (mult, 2H), 3.39 (s, 2H),
3.38 (s, 1H), 3.35 (s, 1H), 3.32 (s, 2H), 3.28 (s, 3H), 3.22 (mult,
1H), 3.10 (mult, 1H), 3.07-2.98 (mult, 2H), 2.92-2.79 (mult,
3H), 2.79 (dd, 0.67H, J ) 16.5, 5.0 Hz), 2.73 (dd, 0.33H, J )
17.5, 3.5 Hz), 2.37-1.03 (comp, 26H), 1.68 (s, 1H), 1.64 (s, 3H),
1.61 (s, 2H), 1.10 (d, 2H, J ) 6.5 Hz), 0.98 (d, 1H, J ) 6.5 Hz),
0.95 (d, 2H, J ) 6.5 Hz), 0.93 (d, 1H, J ) 6.5 Hz), 0.88 (d, 2H,
J ) 6.5 Hz), 0.87 (dd, 3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J )
6.5 Hz); 13C NMR (125 MHz) δ (rotamers) 213.5, 213.4, 196.1,
192.6, 170.8, 169.0, 168.7, 165.8, 164.7, 139.6, 139.0, 138.7,
132.7, 132.2, 129.2, 129.2, 128.8, 128.4, 126.3, 123.3, 123.1,
98.6, 97.1, 82.1, 82.1, 77.9, 76.8, 76.6, 75.2, 73.7, 73.6, 72.9,
72.2, 70.0, 69.0, 68.1, 68.0, 57.5, 57.0, 56.7, 56.6, 56.3, 56.0,
54.9, 54.7, 52.7, 48.7, 48.5, 43.9, 43.5, 43.2, 40.3, 40.1, 39.8,
39.2, 35.7, 35.6, 35.4, 34.6, 34.5, 33.7, 33.0, 32.7, 32.5, 30.4,
29.2, 27.6, 26.3, 26.2, 26.0, 24.6, 24.5, 24.2, 21.1, 20.8, 20.5,
19.5, 16.2, 16.0, 15.8, 15.7, 14.1, 14.0, 11.7, 9.8, 9.5; mass
spectrum (FAB), 953 (M + H)+, 991 (M + K)+. Anal. Calcd
for C53H80N2O13: C, 66.78; H, 8.46; N, 2.94. Found: C, 67.13;
H, 8.33; N, 3.04.
(rotamers 2:1) 8.15 (m, 1H), 7.36-7.25 (m, 5H), 5.34 (d, 0.67H,
J ) 3.0 Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97 (mult, 2H),
4.85 (d, 1H, J ) 2.5 Hz), 4.63-4.56 (comp, 2H), 4.45 (br d,
0.67H, J ) 15.0 Hz), 4.25 (s, 1H), 4.19 (d, 0.67H, J ) 16.5
Hz), 4.18 (d, 0.33H, J ) 16.0 Hz), 3.98 (d, 0.67H, J ) 17.0
Hz), 3.97 (d, 0.33H, J ) 17.5 Hz), 3.98-3.87 (mult, 1.33H),
3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H, J ) 10.0 Hz),
3.59 (mult, 1H), 3.50-3.12 (comp, 4.33H), 3.40 (s, 2H), 3.39
(s, 1H), 3.35 (s, 1H), 3.32 (s, 2H), 3.12 (s, 2H), 3.10 (s, 1H),
3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74
(dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s,
1H), 1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J )
6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz),
0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd,
3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125
MHz) δ (rotamers) 213.2, 213.1, 196.1, 192.7, 170.6, 168.9,
168.6, 165.7, 164.6, 139.5, 138.6, 138.1, 132.6, 132.1, 129.4,
129.2, 128.4, 128.0, 127.2, 123.3, 123.0, 98.6, 97.0, 82.3, 81.8,
77.8, 77.2, 76.6, 75.2, 73.6, 72.8, 72.2, 69.9, 68.9, 68.1, 57.4,
56.9, 56.5, 56.2, 56.1, 55.9, 54.9, 54.7, 52.6, 48.6, 48.3, 43.8,
43.5, 43.4, 43.1, 40.2, 39.7, 39.1, 35.5, 35.5, 35.4, 34.5, 34.4,
33.6, 33.0, 32.6, 32.5, 30.3, 29.2, 27.5, 26.2, 26.1, 26.0, 24.5,
24.4, 24.1, 21.0, 20.7, 20.3, 19.4, 16.1, 15.9, 15.7, 15.6, 14.1,
13.9, 11.6, 9.7, 9.4; mass spectrum (FAB), 977 [M + K]+. Anal.
Calcd for C52H78N2O13: C, 66.50; H, 8.37; N, 2.98. Found: C,
66.74; H, 8.38; N, 2.88.
N-Meth yl-2-p h en eth yla m in e-(32-Ascom ycin yloxya ce-
tic a cid )a m id e (15). N-Methylphenethylamine was coupled
to AOAA (4) on a 4.9 mmol scale according to the method used
for the synthesis of 9. The silica pad (200 g, 230-400 mesh)
was eluted with CH2Cl2 (800 mL) followed by 1% IPA/CH2Cl2
(1 L), 2% IPA/CH2Cl2 (1 L), 3% IPA/CH2Cl2 (1 L), 4% IPA/CH2-
Cl2 (1 L), and then 5% IPA/CH2Cl2 (1 L). Fractions containing
the desired product were combined and concentrated in vacuo
to give 15 (4.4 g, 88% yield): IR (KBr) ν 3440, 3035, 2940,
2880, 2830, 1740, 1700, 1643 cm-1; 1H NMR (500 MHz) CDCl3
δ (rotamers 2:1) 7.34-7.16 (comp, 5H), 5.34 (s, 0.67H), 5.20
(s, 0.33H), 5.09-4.97 (mult, 2H), 4.83 (s, 1H), 4.62 (d, 1H, J )
6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.36 (d, 0.67H, J )
15.0 Hz), 4.32 (d, 0.33H, J ) 15.0 Hz), 4.23 (s, 1H), 4.17 (d,
0.67H, J ) 15.0 Hz), 4.10 (d, 0.33H, J ) 15.0 Hz), 3.98-3.87
(mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H,
J ) 10.0 Hz), 3.50-3.12 (comp, 7.33H), 3.44-3.31 (comp, 9H),
3.03 (comp, 0.67H), 2.97 (s, 1.5H), 2.91 (s, 1.5H), 2.87 (dd, 2H,
J ) 7.5, 7.5 Hz), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74 (dd,
0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s, 1H),
1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J ) 6.0
Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz), 0.92 (d,
1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd, 3H, J )
7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125 MHz) δ
(rotamers) 213.1, 196.0, 192.6, 169.6, 169.4, 168.7, 168.4, 165.6,
164.5, 139.3, 138.8, 138.4, 138.1, 132.0, 131.5, 129.4, 129.2,
128.5, 128.5, 128.4, 128.2, 126.4, 126.0, 123.1, 122.8, 98.4, 96.8,
83.3, 83.2, 82.1, 82.0, 77.5, 77.1, 76.4, 74.9, 73.4, 72.6, 72.0,
69.8, 69.6, 69.3, 68.9, 63.9, 57.2, 57.0, 56.9, 56.8, 56.7, 56.7,
56.3, 56.0, 55.8, 54.7, 54.5, 52.4, 50.5, 49.8, 48.4, 48.1, 43.6,
43.3, 43.0, 39.9, 39.5, 38.9, 36.2, 36.1, 35.2, 34.5, 34.3, 33.4,
33.3, 33.2, 32.7, 32.4, 32.3, 30.5, 29.8, 27.3, 26.0, 26.0, 25.8,
25.1, 24.3, 24.2, 23.9, 20.8, 20.6, 20.2, 19.3, 15.9, 15.7, 15.5,
14.0, 13.8, 11.4, 9.5, 9.2; mass spectrum (FAB), 1005 [M + K]+.
N -Me t h ylb e n zyla m in e -(32-Ascom ycin yloxya ce t ic
a cid )a m id e (13). N-Methylbenzylamine was coupled to
AOAA (4) on a 0.94 mmol scale according to the method used
for the synthesis of 9. The silica plug was eluted with CH2Cl2
(100 mL) followed by 1:1 hexane:acetone (150 mL). Fractions
containing the desired product were combined and concen-
trated in vacuo to give 640 mg of yellow foam which was
further purified by HPLC eluting with 3:2 hexane:acetone to
provide 13 (0.41 g, 45% yield): IR (KBr) ν 3425, 2960 (sh),
2930, 2850, 2820, 1740, 1700, 1643, cm-1; 1H NMR (500 MHz)
CDCl3 δ (rotamers 2:1) 7.39-7.18 (m, 5H), 5.34 (br, 0.67H),
5.20 (br, 0.33H), 5.09-4.97 (mult, 2H), 4.83 (s, 1H), 4.68-4.35
(comp, 5.67H), 4.23 (s, 1H), 3.98-3.87 (mult, 1.33H), 3.75 (br
d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H, J ) 10.0 Hz), 3.59 (mult,
1H), 3.50-2.86 (comp, 5H), 3.43 (s, 2H), 3.42 (s, 1H), 3.39 (s,
1H), 3.35 (s, 2H), 3.33 (s, 2H), 3.31 (s, 1H), 2.79 (m, 0.67H),
2.74 (m, 0.33H), 2.63 (s, 0.25H), 2.37-0.74 (comp, 26H), 2.19
(s, 0.25H), 2.18 (s, 2.5H), 1.67 (s, 1H), 1.64 (s, 1H), 1.62 (s,
2H), 1.60 (s, 2H), 1.01 (d, 2H, J ) 7.0 Hz), 0.97 (d, 1H, J ) 7.0
Hz), 0.95 (d, 2H, J ) 7.5 Hz), 0.93 (d, 1H, J ) 7.5 Hz), 0.88
(dd, 3H, J ) 7.5, 7.5 Hz), 0.87 (d, 2H, J ) 6.5 Hz), 0.84 (d, 1H,
J ) 6.5 Hz); 13C NMR (125 MHz) δ (rotamers) 213.5, 213.4,
196.1, 192.6, 170.1, 170.0, 169.0, 168.7, 165.8, 164.7, 139.7,
138.7, 137.1, 136.8, 132.4, 131.8, 129.6, 128.8, 128.8, 128.5,
128.2, 127.5, 127.4, 126.8, 123.3, 123.1, 98.7, 97.0, 83.5, 83.4,
82.4, 77.8, 77.1, 76.7, 75.2, 73.7, 73.7, 72.9, 72.3, 70.3, 70.1,
70.0, 69.2, 57.5, 57.3, 57.2, 57.0, 56.9, 56.6, 56.3, 56.1, 55.0,
54.7, 53.9, 52.7, 52.6, 51.0, 48.7, 48.5, 43.8, 43.5, 43.1, 40.3,
39.7, 39.2, 36.5, 36.3, 36.2, 35.5, 34.6, 34.6, 33.8, 33.6, 33.5,
32.9, 32.7, 32.6, 30.8, 30.1, 30.0, 29.3, 27.6, 26.4, 26.2, 26.0,
24.6, 24.5, 24.2, 21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8, 15.7,
14.3, 14.2, 11.7, 9.8, 9.4; mass spectrum (FAB), 991 [M + K]+.
Anal. Calcd for C53H80N2O13‚0.5H2O: C, 66.15; H, 8.48; N,
2.91. Found: C, 66.08; H, 8.24; N, 3.07.
Anal. Calcd for C54H82N2O13
: C, 67.05; H, 8.54; N, 2.89.
Found: C, 67.21; H, 8.37; N, 2.93.
Cycloh exyla m in e-(32-Ascom ycin yloxya cet ic a cid )
a m id e (16). Cyclohexylamine was coupled to AOAA (4) on a
0.54 mmol scale according to the method used for the synthesis
of 9. The silica pad was eluted with CH2Cl2 (60 mL) followed
by 2:1 hexane:acetone (200 mL) and then 1:1 hexane:acetone
(100 mL). Fractions containing the desired product were
pooled and concentrated in vacuo to give 0.45 g of white foam
which was further purified by HPLC eluting with 7:4 hexane:
acetone to provide 16 (0.44 g, 86% yield): IR (KBr) ν 3425,
2-P h en eth yla m in e-(32-Ascom ycin yloxya cetic a cid )-
a m id e (14). 2-Phenethylamine was coupled to AOAA (4) on
a 0.71 mmol scale according to the method used for the
synthesis of 9. The silica pad was eluted with CH2Cl2 (100
mL) followed by 1:1 hexane:acetone (125 mL). Fractions
containing the desired product were combined and concen-
trated in vacuo to give 630 mg of yellow foam which was
further purified by HPLC eluting with 3:2 hexane:acetone to
produce 14 (0.45 g, 67% yield): IR (KBr) ν 3435, 1740, 1700,
2920, 2850 (sh), 2860 (sh), 2850, 2820, 1740, 1700, 1643 cm-1
;
1
1650 cm-1; H NMR (500 MHz) CDCl3 δ (rotamers 2:1) 7.82
1H NMR (500 MHz) CDCl3 δ (rotamers 2:1) 7.66 (d, 0.33H, J
) 9.0 Hz), 7.63 (d, 0.67H, J ) 9.0 Hz), 5.34 (d, 0.67H, J ) 3.0
Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97 (mult, 2H), 4.62
(d, 1H, J ) 6.0 Hz), 4.45 (br d, 0.67H, J ) 14.0 Hz), 4.25 (s,
(t, 0.33H, J ) 6.5 Hz), 7.79 (t, 0.67H, J ) 6.5 Hz), 7.30 (dd,
2H, J ) 7.5, 7.5 Hz), 7.22 (d, 2H, J ) 7.5 Hz), 5.33 (d, 0.67H,
J ) 3.7 Hz), 5.22 (d, 0.33H, J ) 3.7 Hz), 5.03 (mult, 2H), 4.85

1772 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Wagner et al.
1H), 4.09 (d, 0.67H, J ) 16.0 Hz), 4.09 (d, 0.33H, J ) 16.0
Hz), 3.98-3.85 (mult, 2.33H), 3.83-3.71 (comp, 1.33H), 3.68
(d, 0.67H, J ) 10.0 Hz), 3.59 (mult, 1H), 3.52-2.97 (comp,
4.33H), 3.45 (s, 3H), 3.40 (s, 2H), 3.39 (s, 1H), 3.35 (s, 1H),
3.31 (s, 2H), 3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5
Hz), 2.74 (dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 36H),
1.67 (s, 1H), 1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d,
2H, J ) 6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5
Hz), 0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88
(dd, 3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR
(125 MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5, 169.6, 169.0,
168.7, 165.8, 164.7, 139.7, 138.7, 132.7, 132.2, 129.2, 123.3,
123.1, 98.7, 97.1, 82.1, 77.8, 77.2, 76.6, 75.2, 73.7, 73.6, 72.9,
72.3, 70.0, 69.0, 67.9, 67.9, 57.5, 57.0, 56.7, 56.6, 56.6, 56.3,
56.0, 55.0, 54.7, 52.7, 48.7, 48.5, 47.7, 43.9, 43.5, 43.2, 40.4,
39.8, 39.2, 35.9, 35.8, 35.5, 34.5, 33.6, 33.0, 32.7, 32.6, 30.4,
29.2, 27.5, 26.3, 26.2, 26.0, 25.6, 24.5, 24.5, 24.2, 21.1, 20.8,
20.5, 19.5, 16.2, 16.0, 15.8, 15.7, 14.2, 14.1, 11.7, 9.8, 9.5; mass
spectrum (FAB), 969 [M + K]+, 931 [M + H]+. Anal. Calcd
for C51H82N2O13: C, 65.78; H, 8.88; N, 3.01. Found: C, 65.60;
H, 8.70; N, 2.93.
Hz), 4.25 (br s, 1H), 4.03 (d, 0.67H, J ) 16.0 Hz), 4.02 (d, 0.33H,
J ) 16.0 Hz), 3.98-3.87 (mult, 1.33H), 3.75 (br d, 0.33H, J )
14.5 Hz), 3.68 (d, 0.67H, J ) 10.0 Hz), 3.58 (s, 3H), 3.57 (mult,
1H), 3.50-3.09 (comp, 4.33H), 3.39 (s, 3H), 3.38 (s, 1H), 3.35
(s, 1H), 3.31 (s, 2H), 3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J )
17.5, 4.5 Hz), 2.74 (dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74
(comp, 26H), 1.67 (s, 1H), 1.65 (s, 2H), 1.64 (s, 1H), 1.61 (s,
2H), 1.01 (d, 2H, J ) 6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d,
2H, J ) 7.5 Hz), 0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5
Hz), 0.88 (dd, 3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz);
13C NMR (125 MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5,
169.9, 169.0, 168.7, 165.8, 164.7, 145.3, 141.2, 139.7, 138.8,
134.8, 134.8, 133.0, 132.4, 129.0, 128.9, 126.9, 123.6, 123.3,
123.1, 98.7, 97.1, 82.3, 82.2, 77.8, 77.1, 76.6, 75.2, 73.7, 73.6,
72.9, 72.2, 70.0, 68.9, 67.9, 57.5, 57.0, 56.9, 56.6, 56.3, 56.0,
55.0, 54.7, 52.7, 48.7, 48.5, 43.9, 43.5, 43.1, 40.4, 39.8, 39.2,
35.7, 35.6, 34.6, 34.5, 33.7, 32.9, 32.7, 32.6, 30.4, 29.0, 27.5,
26.3, 26.2, 26.0, 24.6, 24.5, 24.2, 21.2, 20.8, 20.5, 19.5, 16.2,
16.0, 15.8, 15.6, 14.3, 14.2, 11.7, 9.8, 9.5; mass spectrum (FAB),
964 [M + K]+, 926 [M + H]+. Anal. Calcd for C50H75N3O13
C, 64.84; H, 8.16; N, 4.54. Found: C, 64.98; H, 8.22; N, 4.36.
4-Am in op yr id in e-(32-Ascom ycin yloxya cet ic a cid )
:
2-Am in op yr id in e-(32-Ascom ycin yloxya cet ic
a cid )
a m id e (17). 2-Aminopyridine was coupled to AOAA (4) on a
0.94 mmol scale according to the method used for the synthesis
of 9. The silica pad was eluted with CH2Cl2 (100 mL) followed
by 2:1 hexane:acetone (250 mL). Fractions containing the
desired product were combined and concentrated in vacuo to
give 0.42 g of yellow foam which was further purified by HPLC
eluting with 2:1 hexane:acetone to afford 17 (0.15 g, 17%
yield): IR (KBr) ν 3425, 3398 (sh), 3250 (sh), 2960 (sh), 2938,
a m id e (19). 4-Aminopyridine was coupled to AOAA (4) on a
0.94 mmol scale according to the method used for the synthesis
of 9. The silica pad was eluted with CH2Cl2 (60 mL) followed
by 1:1 hexane:acetone (150 mL) and then 1:2 hexane:acetone
(150 mL). Fractions containing the desired product were
combined and concentrated in vacuo to give 0.53 g of yellow
foam which was further purified by HPLC eluting with 1:1
hexane:acetone to provide 19 (0.21 g, 25% yield): IR (KBr) ν
3425, 3310 (sh), 2960 (sh), 2930, 2850, 2820, 1740, 1700, 1643,
2850, 2820, 1740, 1700, 1643, 1595(sh) cm-1 1H NMR (500
;
1
MHz) CDCl3 δ (rotamers 2:1) 10.04 (s, 1H), 8.33 (d, 1H, J )
3.5 Hz), 8.26 (d, 1H, J ) 9.5 Hz), 7.70 (ddd, 1H, J ) 7.5, 7.5,
2.0 Hz), 7.04 (ddd, 1H, J ) 7.5, 7.5, 5.5 Hz), 5.35 (d, 0.67H, J
) 3.0 Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97 (mult, 2H),
4.87 (s, 1H), 4.62 (d, 1H, J ) 6.0 Hz), 4.45 (br d, 0.67H, J )
15.0 Hz), 4.29 (d, 0.67H, J ) 16.0 Hz), 4.28 (d, 0.33H, J )
16.0 Hz), 4.24 (br s, 1H), 4.05 (d, 1H, J ) 16.0 Hz), 3.98-3.87
(mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H,
J ) 10.0 Hz), 3.58 (s, 3H), 3.57 (mult, 1H), 3.50-3.09 (comp,
4.33H), 3.39 (s, 3H), 3.38 (s, 1H), 3.35 (s, 1H), 3.31 (s, 2H),
3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74
(dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s,
1H), 1.65 (s, 2H), 1.64 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J )
6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz),
0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd,
3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125
MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5, 169.9, 169.0,
168.7, 165.8, 164.7, 151.4, 148.2, 139.7, 138.7, 138.0, 132.7,
132.2, 129.2, 123.4, 123.1, 119.7, 114.1, 98.7, 97.1, 83.4, 82.2,
77.8, 77.1, 76.6, 75.2, 73.7, 73.6, 72.9, 72.2, 70.0, 69.2, 69.1,
57.5, 57.0, 56.9, 56.6, 56.3, 56.0, 55.0, 54.7, 52.7, 48.7, 48.5,
43.9, 43.5, 43.1, 40.4, 39.8, 39.2, 35.9, 35.7, 34.6, 34.5, 33.7,
32.9, 32.7, 32.6, 30.4, 29.7, 27.5, 26.3, 26.2, 26.0, 24.6, 24.5,
24.2, 21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8, 15.6, 14.3, 14.2,
11.7, 9.8, 9.5; mass spectrum (FAB), 964 [M + K]+. Anal.
Calcd for C50H75N3O13: C, 64.84; H, 8.16; N, 4.54. Found: C,
65.10; H, 8.13; N, 4.78.
1595 cm-1; H NMR (500 MHz) CDCl3 δ (rotamers 2:1) 9.84
(s, 0.33H), 9.82 (s, 0.67H), 8.53 (d, 2H, J ) 6.0 Hz), 7.54 (d,
2H, J ) 5.5 Hz), 5.34 (d, 0.67H, J ) 3.0 Hz), 5.22 (d, 0.33H, J
) 3.0 Hz), 5.09-4.97 (mult, 2H), 4.87 (s, 1H), 4.62 (d, 1H, J )
6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.28 (d, 0.67H, J )
16.0 Hz), 4.27 (d, 0.33H, J ) 16.0 Hz), 4.25 (br s, 1H), 4.03 (d,
0.67H, J ) 16.0 Hz), 4.02 (d, 0.33H, J ) 16.0 Hz), 4.00-3.87
(mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H,
J ) 10.0 Hz), 3.62-3.08 (comp, 5.33H), 3.58 (s, 3H), 3.39 (s,
3H), 3.35 (s, 1H), 3.31 (s, 2H), 3.01 (comp, 0.67H), 2.79 (dd,
0.67H, J ) 17.5, 4.5 Hz), 2.74 (dd, 0.33H, J ) 17.5, 4.5 Hz),
2.40-0.74 (comp, 26H), 1.70 (s, 1H), 1.67 (s, 2H), 1.63 (s, 1H),
1.61 (s, 2H), 1.01 (d, 2H, J ) 6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz),
0.95 (d, 2H, J ) 7.5 Hz), 0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H,
J ) 7.5 Hz), 0.88 (dd, 3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J )
7.5 Hz); 13C NMR (125 MHz) δ (rotamers) 213.5, 213.4, 196.1,
192.5, 170.3, 169.0, 168.7, 165.8, 164.7, 150.7 144.8, 139.7,
138.8, 133.0, 132.5, 129.0, 128.9, 123.3, 123.1, 113.7, 98.7, 97.1,
82.3, 82.2, 77.8, 77.1, 76.6, 75.2, 73.7, 73.6, 72.9, 72.2, 70.0,
68.9, 67.9, 57.5, 57.0, 56.9, 56.6, 56.3, 56.0, 55.0, 54.7, 52.7,
48.7, 48.5, 43.9, 43.5, 43.1, 40.4, 39.8, 39.2, 35.7, 35.6, 35.5,
34.6, 34.5, 33.7, 32.9, 32.7, 32.6, 30.4, 29.0, 27.5, 26.3, 26.2,
26.0, 24.6, 24.5, 24.2, 21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8,
15.6, 14.3, 14.2, 11.7, 9.8, 9.5; mass spectrum (FAB), 964 [M
+ K]+. Anal. Calcd for C50H75N3O13: C, 64.84; H, 8.16; N,
4.54. Found: C, 65.15; H, 8.30; N, 4.27.
4-Am in oph en ol-(32-Ascom ycin yloxyacetic acid)am ide
(20). 4-Aminophenol was coupled to AOAA (4) on a 0.94 mmol
scale according to the method used for the synthesis of 9. The
silica pad was eluted with CH2Cl2 (150 mL) followed by 3:1
hexane:acetone (100 mL) and then 2:1 hexane:acetone (200
mL). Fractions containing the desired product were combined
and concentrated in vacuo to give 0.58 g tan foam which was
further purified by HPLC eluting with 3:2 hexane:acetone to
provide 20 (0.29 g, 33% yield): IR (KBr) ν 3425, 3275 2960
3-Am in op yr id in e-(32-Ascom ycin yloxya cet ic
a cid )
a m id e (18). 3-Aminopyridine was coupled to AOAA (4) on a
0.94 mmol scale according to the method used for the synthesis
of 9. The silica pad was eluted with CH2Cl2 (60 mL) followed
by 1:1 hexane:acetone (150 mL) and then 1:2 hexane:acetone
(100 mL). Fractions containing the desired product were
combined and concentrated in vacuo to give 0.55 g of yellow
foam which was further purified by HPLC eluting with 1:1
hexane:acetone to provide 18 (0.42 g, 48% yield): IR (KBr) ν
3425, 3310 (sh), 2960 (sh), 2930, 2850, 2820, 1740, 1700, 1643,
1
(sh), 2938, 2850, 2820, 1740, 1700, 1643 cm-1; H NMR (500
MHz) CDCl3 δ (rotamers 2:1) 9.51 (s, 0.33H), 9.50 (s, 0.67H),
7.48 (d, 2H, J ) 9.5 Hz), 6.81 (d, 2H, J ) 9.5 Hz), 5.34 (d,
0.67H, J ) 3.0 Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97
(mult, 2H), 4.94 (br s, 2H), 4.87 (d, 1H, J ) 2.5 Hz), 4.62 (d,
1H, J ) 6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.23 (d, 1H,
J ) 15.5 Hz), 4.01 (d, 0.67H, J ) 17.0 Hz), 4.00 (d, 0.33H, J )
17.0 Hz), 3.98-3.87 (mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5
1
1595 cm-1; H NMR (500 MHz) CDCl3 δ (rotamers 2:1) 9.82
(s, 1H), 8.62 (m, 1H), 8.37 (dd, 1H, J ) 4.5, 2.0 Hz), 8.29 (comp
dd, 1H, J ) 9.0, 9.0 Hz), 7.29 (m, 1H), 5.39 (d, 0.67H, J ) 3.0
Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.19-4.97 (mult, 2H), 4.89
(s, 1H), 4.62 (d, 1H, J ) 6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0
Hz), 4.28 (d, 0.67H, J ) 16.0 Hz), 4.27 (d, 0.33H, J ) 16.0

Immunophilin Binding and Immunosuppressive Potency
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1773
Hz), 3.68 (d, 0.67H, J ) 10.0 Hz), 3.50-3.09 (comp, 4.33H),
3.54 (s, 3H), 3.39 (s, 2H), 3.38 (s, 1H), 3.35 (s, 1H), 3.31 (s,
2H), 3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz),
2.74 (dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.69
(s, 1H), 1.67 (s, 2H), 1.64 (s, 1H), 1.56 (s, 2H), 1.01 (d, 2H, J )
6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz),
0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd,
3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125
MHz) δ (rotamers) 213.6, 213.5, 196.1, 192.6, 169.0, 169.0,
168.7, 165.8, 164.7, 152.9, 139.6, 138.8, 132.8, 132.2, 130.7,
129.2, 129.1, 123.3, 123.1, 121.7, 115.6, 98.7, 97.1, 82.2, 82.1,
77.8, 77.3, 76.6, 75.3, 73.7, 73.6, 72.9, 72.3, 70.0, 69.0, 68.0,
57.5, 57.0, 56.8, 56.6, 56.3, 56.0, 55.0, 54.7, 52.7, 48.7, 48.5,
43.9, 43.5, 43.3, 40.4, 39.8, 39.2, 35.7, 35.6, 35.5, 34.6, 34.5,
33.7, 33.0, 32.7, 32.6, 31.6, 30.5, 29.3, 29.1, 27.5, 26.3, 26.2,
26.0, 24.6, 24.5, 24.2, 22.6, 21.1, 20.8, 20.4, 19.5, 16.2, 16.0,
15.8, 15.7, 14.2, 14.1, 11.7, 9.8, 9.5; mass spectrum (FAB), 979
[M + K]+. Anal. Calcd for C51H76N2O14: C, 65.08; H, 8.13; N,
2.97. Found: C, 64.80; H, 8.10; N, 2.89.
solution of 1 N H3PO4 (75 mL) and EtOAc (75 mL). The
organic layer was washed with saturated NaHCO3 solution
(75 mL) and brine (75 mL), dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was dissolved in CH2Cl2
and then passed through a pad of silica gel (50 g) eluting with
CH2Cl2 (200 mL), 6:1 CH2Cl2:CH3CN (200 mL), 4:1 CH2Cl2:
CH3CN (100 mL), and then 1:1 CH2Cl2:CH3CN (100 mL),
collecting 20 mL fractions throughout. Eluate containing the
desired product was concentrated in vacuo to give 0.5 g of
yellowish-brown foam, which was further purified by HPLC
eluting with 4:1 CH2Cl2:CH3CN to provide 22 (0.3 g, 42%
yield): IR (KBr) ν 3440, 3280, 3100, 2960 (sh), 2920, 2865,
2820, 1740, 1700, 1640, 1580, 1520 cm-1; 1H NMR (500 MHz)
CDCl3 δ (rotamers 2:1) 9.74 (s, 0.33H), 9.73 (s, 0.67H), 7.79
(dd, 1H, J ) 3.0, 3.0 Hz), 7.49 (comp, 1H), 7.39 (d, 1H, J ) 9.5
Hz), 5.34 (d, 0.67H, J ) 3.0 Hz), 5.22 (d, 0.33H, J ) 3.0 Hz),
5.09-4.97 (mult, 2H), 4.88 (d, 1H, J ) 2.5 Hz), 4.62 (d, 1H, J
) 6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.25 (s, 1H), 4.24
(d, 0.67H, J ) 16.5 Hz), 4.23 (d, 0.33H, J ) 16.0 Hz), 3.99 (d,
0.67H, J ) 17.0 Hz), 3.98 (d, 0.33H, J ) 17.5 Hz), 3.98-3.87
(mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H,
J ) 10.0 Hz), 3.59 (mult, 1H), 3.56 (s, 3H), 3.50-3.12 (comp,
4.33H), 3.40 (s, 2H), 3.39 (s, 1H), 3.35 (s, 1H), 3.30 (s, 2H),
3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74
(dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s,
1H), 1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J )
6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz),
0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd,
3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125
MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5, 169.4, 169.0,
168.7, 165.8, 164.7, 139.7, 138.8, 137.5, 133.0, 132.6, 132.4,
130.5, 128.9, 128.8, 123.3, 123.1, 121.4, 119.0, 98.7, 97.1, 82.3,
82.2, 77.8, 77.1, 76.6, 75.2, 73.7, 73.6, 72.9, 72.3, 70.0, 69.0,
67.8, 57.5, 57.0, 56.8, 56.6, 56.3, 56.0, 55.0, 54.7, 52.7, 48.7,
48.5, 43.9, 43.5, 43.1, 40.4, 39.8, 39.2, 35.6, 35.5, 34.6, 34.5,
33.7, 33.0, 32.7, 32.6, 30.4, 29.0, 27.5, 26.4, 26.2, 26.0, 24.6,
24.5, 24.2, 21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.9, 15.7, 14.2,
14.1, 11.7, 9.8, 9.5; mass spectrum (FAB), 1031 [M + K]+. Anal.
Calcd for C51H74N2O13Cl2: C, 61.62; H, 7.50; N, 2.81. Found:
C, 62.02; H, 7.54; N, 2.84.
4-Mor ph olin oa n ilin e-(32-Ascom ycin yloxya cetic a cid )-
a m id e (21). To a stirring solution of AOAA (4) (0.75 g, 0.88
mmol) in CH2Cl2 (10 mL) at 0 °C were added DMAP (0.02 g,
0.18 mmol) and 4-morpholinoaniline (0.31 g, 1.76 mmol)
followed by 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide
hydrochloride (EDAC) (0.20 g, 1.1 mmol). The reaction was
stirred overnight at room temperature then partitioned be-
tween EtOAc (150 mL) and 1 N HCl (150 mL). The organic
layer was washed with an additional portion of 1 N HCl (150
mL), saturated NaHCO3 solution (2 × 150 mL), and then brine
(150 mL). The organic layer was dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was dissolved in CH2Cl2
and then passed through a pad of silica gel (20 g) eluting with
CH2Cl2 (150 mL), 2:1 hexane:acetone (100 mL), 6:4 hexane:
acetone (100 mL), and then finally 1:1 hexane:acetone (100
mL), collecting 20 mL fractions throughout. The eluate
containing the desired product was pooled and concentrated
in vacuo to give 0.64 g of yellow foam. This material was
further purified by HPLC eluting with 4:5 CH2Cl2:CH3CN to
provide 21 (0.43 g, 48% yield): IR (KBr) ν 3425, 3310 (sh),
2960 (sh), 2930, 2850, 2820, 1740, 1700, 1670 (sh), 1643, 1595
cm-1; 1H NMR (500 MHz) CDCl3 δ (rotamers 2:1) 9.49 (br, 1H),
7.53 (d, 2H, J ) 9.5 Hz), 6.91 (br, 2H), 5.34 (d, 0.67H, J ) 3.0
Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97 (mult, 2H), 4.86
(d, 1H, J ) 1.0 Hz), 4.62 (d, 1H, J ) 6.0 Hz), 4.45 (br d, 0.67H,
J ) 15.0 Hz), 4.23 (d, 1H, J ) 17.0 Hz), 4.21 (s, 1H), 4.01 (d,
0.67H, J ) 17.5 Hz), 4.00 (d, 0.33H, J ) 17.5 Hz), 3.98-3.82
(mult, 5.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H,
J ) 10.0 Hz), 3.59 (mult, 1H), 3.60-3.07 (comp, 8.33H), 3.55
(s, 3H), 3.39 (s, 2H), 3.38 (s, 1H), 3.35 (s, 1H), 3.31 (s, 2H),
3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74
(dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.69 (s,
1H), 1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J )
6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz),
0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd,
3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125
MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5, 169.0, 168.7,
165.8, 164.7, 148.1, 139.7, 138.7, 132.9, 132.3, 130.9, 129.1,
129.0, 123.3, 123.1, 120.9, 116.3, 98.7, 97.1, 82.2, 82.2, 77.8,
77.1, 76.6, 75.2, 73.7, 73.6, 72.9, 72.2, 70.0, 69.0, 68.0, 66.9,
57.5, 57.0, 56.8, 56.6, 56.3, 56.0, 55.0, 54.7, 52.7, 49.9, 48.7,
48.5, 43.9, 43.5, 43.1, 40.3, 39.8, 39.2, 35.7, 35.6, 35.5, 34.6,
34.5, 33.6, 32.9, 32.7, 32.6, 30.4, 29.1, 27.5, 26.3, 26.2, 26.0,
24.6, 24.5, 24.2, 21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8, 15.7,
14.3, 14.2, 11.7, 9.8, 9.5; mass spectrum (FAB), 1048 [M + K]+,
1009 [M]+. Anal. Calcd for C55H83N3O14: C, 65.39; H, 8.28;
N, 4.16. Found: C, 65.67; H, 8.32; N, 3.96.
4-Ch lor oa n ilin e-(32-Ascom ycin yloxya cet ic
a cid )
a m id e (23). 4-Chloroaniline was coupled to AOAA (4) on a
0.59 mmol scale according to the method used for the synthesis
of 9. The silica pad was eluted with CH2Cl2 (100 mL) followed
by 1:1 hexane:acetone (250 mL). Fractions containing the
desired product were combined and concentrated in vacuo to
give 0.49 g of yellow foam which was further purified by HPLC
eluting with 5:2 hexane:acetone to provide 23 (0.42 g, 74%
yield): IR (KBr) ν 3440, 3300 (sh), 3120, 3060, 2960 (sh), 2930,
2865, 2820, 1740, 1695, 1650, 1595 cm-1; 1H NMR (500 MHz)
CDCl3 δ (rotamers 2:1) 9.66 (s, 0.33H), 9.65 (s, 0.67H), 7.57
(dd, 2H, J ) 10.0, 3.0 Hz), 7.30 (dd, 2H, J ) 9.5, 3.0 Hz), 5.34
(d, 0.67H, J ) 3.0 Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97
(mult, 2H), 4.88 (d, 1H, J ) 2.5 Hz), 4.62 (d, 1H, J ) 6.0 Hz),
4.45 (br d, 0.67H, J ) 15.0 Hz), 4.26 (s, 1H), 4.24 (d, 0.67H, J
) 16.5 Hz), 4.23 (d, 0.33H, J ) 16.0 Hz), 4.00 (d, 0.67H, J )
17.0 Hz), 3.99 (d, 0.33H, J ) 17.5 Hz), 3.98-3.87 (mult, 1.33H),
3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H, J ) 10.0 Hz),
3.59 (mult, 1H), 3.50-3.12 (comp, 4.33H), 3.55 (s, 3H), 3.39
(s, 2H), 3.38 (s, 1H), 3.35 (s, 1H), 3.30 (s, 2H), 3.01 (comp,
0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74 (dd, 0.33H, J
) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s, 1H), 1.65 (s,
2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J ) 6.0 Hz), 0.98
(d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz), 0.92 (d, 1H, J )
7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd, 3H, J ) 7.5, 7.5
Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125 MHz) δ (rotamers)
213.5, 213.4, 196.1, 192.5, 169.2, 169.0, 168.7, 165.8, 164.7,
139.7, 138.8, 136.6, 133.0, 132.4, 129.1, 128.9, 123.3, 123.1,
121.0, 98.7, 97.1, 82.2, 82.2, 77.8, 77.1, 76.5, 75.2, 73.7, 73.6,
72.9, 72.3, 70.0, 69.0, 67.9, 57.5, 57.0, 56.8, 56.6, 56.3, 56.0,
55.0, 54.7, 52.7, 48.7, 48.5, 43.9, 43.5, 43.1, 40.4, 39.8, 39.2,
35.6, 35.6, 35.5, 34.6, 34.5, 33.7, 33.0, 32.7, 32.6, 30.4, 29.0,
27.5, 26.4, 26.2, 26.0, 24.6, 24.5, 24.2, 21.2, 20.8, 20.5, 19.5,
16.2, 16.0, 15.9, 15.7, 14.2, 14.1, 11.7, 9.8, 9.5; mass spectrum
3,4-Dich lor oa n ilin e-(32-Ascom ycin yloxya cetic a cid )-
a m id e (22). Ethylmagnesium bromide (1 M in THF, 9.6 mL)
was cooled to 0 °C, to which was added in a dropwise fashion
a solution of 3,4-dichloroaniline (1.6 g, 9.6 mmol) in dry THF
(1.5 mL). The mixture was stirred for 15 min and cooled to
-78 °C, whereupon a solution of AOAA benzyl ester (3b) (0.8
g, 0.8 mmol) in dry THF (2 mL) was added dropwise. After 3
h the mixture was added dropwise to a stirring biphasic

1774 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Wagner et al.
(FAB), 997 [M + K]+. Anal. Calcd for C51H75N2O13Cl: C,
63.83; H, 7.87; N, 2.91. Found: C, 63.72; H, 7.75; N, 2.91.
1031 [M + K]+. Anal. Calcd for C52H75N2O13F3: C, 62.89; H,
7.61; N, 2.82. Found: C, 62.49; H, 7.31; N, 2.78.
4-Met h oxya n ilin e-(32-Ascom ycin yloxya cet ic a cid )
a m id e (24). 4-Methoxyaniline was coupled to AOAA (4) on a
0.59 mmol scale according to the method used for the synthesis
of 9. The silica pad was eluted with CH2Cl2 (150 mL) followed
by 2:1 hexane:acetone (300 mL). Fractions containing the
desired product were combined and concentrated in vacuo to
give 0.46 g of yellow foam which was further purified by HPLC
eluting with 2:1 hexane:acetone to afford 24 (0.27 g, 48%
yield): IR (KBr) ν 3440, 3305 (sh), 2960 (sh), 2930, 2865, 2820,
4-Am in ob en zot r iflu or id e-(32-Ascom ycin yloxya cet ic
a cid )a m id e (26). 4-Aminobenzotrifluoride was coupled to
AOAA (4) on a 0.74 mmol scale according to the method used
for the synthesis of 21. The silica pad (40 g, 70-230 mesh)
was eluted with CH2Cl2 (300 mL) followed by 2:1 hexane:
acetone (300 mL). Fractions containing the desired product
were combined and concentrated in vacuo to give 0.77 g of pale
green foam which was further purified by HPLC eluting with
5:2 hexane:acetone to give 0.66 g of a semipure material.
Further purification by HPLC eluting with 3:1 CH2Cl2:
CH3CN produced pure 26 (0.50 g, 68% yield): IR (KBr) ν 3425,
3280 (sh), 2960 (sh), 2920, 2850, 2820, 1740, 1700, 1643, 1620
1740, 1695, 1650, 1600 cm-1 1H NMR (500 MHz) CDCl3
; δ
(rotamers 2:1) 9.51 (s, 0.33H), 9.50 (s, 0.33H), 5.34 (d, 0.67H,
J ) 3.0 Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97 (mult, 2H),
4.88 (d, 1H, J ) 2.5 Hz), 4.62 (d, 1H, J ) 6.0 Hz), 4.45 (br d,
0.67H, J ) 15.0 Hz), 4.25 (s, 1H), 4.23 (d, 0.67H, J ) 16.5
Hz), 4.22 (d, 0.33H, J ) 16.0 Hz), 4.00 (d, 0.67H, J ) 17.0
Hz), 3.99 (d, 0.33H, J ) 17.5 Hz), 3.98-3.87 (mult, 1.33H),
3.81 (s, 3H), 3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H, J
) 10.0 Hz), 3.59 (mult, 1H), 3.55 (s, 3H), 3.50-3.12 (comp,
4.33H), 3.39 (s, 2H), 3.38 (s, 1H), 3.35 (s, 1H), 3.31 (s, 2H),
3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74
(dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 1.67 (s,
1H), 1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J )
6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz),
0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd,
3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125
MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5, 169.0, 168.7,
165.8, 164.7, 156.3, 139.7, 138.7, 132.9, 132.3, 131.2, 129.0,
129.0, 123.3, 123.0, 121.3, 114.1, 98.7, 97.1, 82.2, 82.2, 77.8,
77.1, 76.6, 75.2, 73.7, 73.6, 72.9, 72.2, 70.0, 69.0, 68.0, 57.5,
57.0, 56.9, 56.6, 56.3, 56.0, 55.5, 55.0, 54.7, 52.7, 48.7, 48.5,
43.9, 43.5, 43.1, 40.3, 39.8, 39.2, 35.7, 35.6, 35.5, 34.6, 34.5,
33.6, 32.9, 32.7, 32.6, 30.4, 29.1, 27.5, 26.3, 26.2, 26.0, 24.6,
24.5, 24.2, 21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8, 15.7, 14.2,
14.1, 11.7, 9.8, 9.4; mass spectrum (FAB), 993 [M + K]+. Anal.
Calcd for C52H78N2O14: C, 65.38; H, 8.23; N, 2.93. Found: C,
65.28; H, 8.13; N, 2.92.
1
(sh), 1600 cm-1; H NMR (500 MHz) CDCl3 δ (rotamers 2:1)
9.83 (s, 0.33H), 9.81 (s, 0.67H), 7.74 (d, 2H, J ) 8.5 Hz), 7.59
(d, 2H, J ) 8.5 Hz), 5.34 (d, 0.67H, J ) 3.0 Hz), 5.22 (d, 0.33H,
J ) 3.0 Hz), 5.09-4.97 (mult, 2H), 4.88 (d, 1H, J ) 2.5 Hz),
4.62 (d, 1H, J ) 6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.27
(d, 0.67H, J ) 16.5 Hz), 4.26 (d, 0.33H, J ) 16.0 Hz), 4.24 (br
s, 1H), 4.02 (d, 0.67H, J ) 17.0 Hz), 4.01 (d, 0.33H, J ) 17.5
Hz), 3.98-3.87 (mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz),
3.68 (d, 0.67H, J ) 10.0 Hz), 3.59 (mult, 1H), 3.54-3.08 (comp,
4.33H), 3.57 (s, 3H), 3.39 (s, 2H), 3.38 (s, 1H), 3.35 (s, 1H),
3.31 (s, 2H), 3.01 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 16.0, 4.0
Hz), 2.74 (dd, 0.33H, J ) 16.0, 4.0 Hz), 2.37-0.74 (comp, 26H),
1.70 (s, 1H), 1.67 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d,
2H, J ) 6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5
Hz), 0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88
(dd, 3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR
(125 MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5, 169.6, 169.0,
168.7, 165.8, 164.7, 141.0, 139.7, 138.8, 133.0, 132.4, 128.9,
128.9, 126.6, 126.4, 126.2, 126.2, 125.9, 125.6, 125.2, 123.3,
123.1, 119.4, 98.7, 97.1, 82.3, 82.2, 77.9, 77.2, 76.6, 75.2, 73.7,
73.6, 72.9, 72.3, 70.0, 68.9, 67.9, 57.6, 57.0, 56.8, 56.6,
56.3, 56.0, 55.0, 54.7, 52.7, 48.7, 48.5, 43.9, 43.5, 43.1,
40.4, 39.8, 39.2, 35.6, 35.5, 35.5, 34.6, 34.5, 33.7, 33.0, 32.7,
32.6, 30.4, 29.0, 27.5, 26.4, 26.2, 26.0, 24.6, 24.5, 24.2,
21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.9, 15.7, 14.3, 14.2, 11.7,
9.8, 9.5; mass spectrum (FAB), 1031 [M + K]+. Anal. Calcd
for C52H75N2O13F3: C, 62.89; H, 7.61; N, 2.82. Found: C, 63.29;
H, 7.69; N, 2.82.
3-Am in ob en zot r iflu or id e-(32-Ascom ycin yloxya cet ic
a cid )a m id e (25). 3-Aminobenzotrifluoride was coupled to
AOAA (4) on a 0.59 mmol scale according to the method used
for the synthesis of 9. The silica pad was eluted with CH2Cl2
(50 mL) followed by 4:1 hexane:acetone (250 mL), 3:1 hexane:
acetone (100 mL), and then finally 1:1 hexane:acetone (150
mL). Fractions containing the desired product were combined
and concentrated in vacuo to give 0.57 g of yellow foam
which was further purified by HPLC eluting with 4:1 CH2Cl2:
CH3CN to provide 25 (0.39 g, 67% yield): IR (KBr) ν 3425,
3290, 2920, 2860, 2820, 1740, 1700, 1645, 1620 (sh), 1601, 1550
3-F lu or oa n ilin e-(32-Ascom ycin yloxya cet ic
a cid )
a m id e (27). 3-Fluoroaniline was coupled to AOAA (4) on a
0.59 mmol scale according to the method used for the synthesis
of 9. The silica pad was eluted with CH2Cl2 (60 mL) followed
by 3:1 hexane:acetone (250 mL). Fractions containing the
desired product were combined and concentrated in vacuo to
give 0.55 g of white foam which was further purified by HPLC
eluting with 5:2 hexane:acetone to provide 27 (0.47 g, 85%
yield): IR (KBr) ν 3420, 3290, 2960 (sh), 2940, 2880, 2820,
1
cm-1; H NMR (500 MHz) CDCl3 δ (rotamers 2:1) 10.84 (m,
1H), 7.94 (dd, 1H, J ) 9.0, 9.0 Hz), 7.80 (comp, 1H), 7.46 (dd,
1H, J ) 9.0, 9.0 Hz), 7.37 (d, 1H, J ) 9.0), 5.34 (d, 0.67H, J )
3.0 Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97 (mult, 2H),
4.62 (d, 1H, J ) 5.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.25
(s, 1H), 4.26 (d, 0.67H, J ) 17.5 Hz), 4.25 (d, 0.33H, J ) 17.5
Hz), 4.02 (d, 0.67H, J ) 17.5 Hz), 4.01 (d, 0.33H, J ) 17.5
Hz), 3.98-3.87 (mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz),
3.68 (d, 0.67H, J ) 10.0 Hz), 3.61-3.06 (comp, 5.33H), 3.57
(s, 3H), 3.39 (s, 2H), 3.38 (s, 1H), 3.35 (s, 1H), 3.31 (s, 2H),
3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 15.5, 3.0 Hz), 2.74
(dd, 0.33H, J ) 15.5, 3.0 Hz), 2.37-0.74 (comp, 26H), 1.67 (s,
1H), 1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01 (d, 2H, J )
6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5 Hz),
0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88 (dd,
3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR (125
MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5, 169.5, 169.0,
168.7, 165.8, 164.7, 139.7, 138.8, 138.5, 133.0, 132.4, 131.7,
131.4, 131.1, 130.8, 129.5, 128.9, 128.9, 123.3, 123.1, 122.8,
120.6, 116.4, 98.7, 97.1, 82.3, 82.1, 77.8, 77.1, 76.6, 75.2, 73.7,
73.6, 72.9, 72.3, 70.0, 69.0, 67.8, 57.5, 57.0, 56.6, 56.3, 56.0,
55.0, 54.7, 52.7, 48.7, 48.5, 43.9, 43.5, 43.1, 40.3, 39.8, 39.2,
35.6, 35.5, 34.6, 34.5, 33.7, 32.9, 32.7, 32.6, 30.4, 29.0, 27.5,
26.4, 26.2, 26.0, 24.6, 24.6, 24.2, 21.2, 20.8, 20.5, 19.5, 16.2,
16.0, 15.9, 15.7, 14.3, 14.2, 11.7, 9.8, 9.5; mass spectrum (FAB),
1
1740, 1695 (sh), 1690, 1645, 1610, 1540 cm-1; H NMR (500
MHz) CDCl3 δ (rotamers 2:1) 9.70 (s, 0.33H), 9.69 (s, 0.67H),
7.52 (ddd, 1H, J ) 11.0, 1.0, 1.0 Hz), 7.29 (comp, 2H), 6.82
(dddd, 1H, J ) 8.0, 8.0, 1.0, 1.0 Hz), 5.34 (d, 0.67H, J ) 3.0
Hz), 5.22 (d, 0.33H, J ) 3.0 Hz), 5.09-4.97 (mult, 2H), 4.88
(d, 1H, J ) 2.5 Hz), 4.62 (d, 1H, J ) 5.0 Hz), 4.45 (br d, 0.67H,
J ) 12.5 Hz), 4.25 (s, 1H), 4.25 (d, 0.67H, J ) 16.0 Hz), 4.24
(d, 0.33H, J ) 16.0 Hz), 4.02 (d, 0.67H, J ) 16.0 Hz), 4.01 (d,
0.33H, J ) 16.0 Hz), 3.98-3.87 (mult, 1.33H), 3.75 (br d,
0.33H, J ) 13.5 Hz), 3.68 (d, 0.67H, J ) 10.0 Hz), 3.59 (mult,
1H), 3.57 (s, 3H), 3.54-3.06 (comp, 4.33H), 3.39 (s, 2H), 3.38
(s, 1H), 3.35 (s, 1H), 3.31 (s, 2H), 3.02 (comp, 0.67H), 2.79 (dd,
0.67H, J ) 15.5, 3.5 Hz), 2.74 (dd, 0.33H, J ) 15.5, 3.5 Hz),
2.37-0.74 (comp, 26H), 1.70 (s, 1H), 1.67 (s, 2H), 1.64 (s, 1H),
1.61 (s, 2H), 1.01 (d, 2H, J ) 6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz),
0.95 (d, 2H, J ) 7.5 Hz), 0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H,
J ) 7.5 Hz), 0.88 (dd, 3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J )
7.5 Hz); 13C NMR (125 MHz) δ (rotamers) 213.5, 213.4, 196.1,
192.5, 169.3, 169.0, 168.7, 165.8, 164.7, 164.0, 162.0, 139.7,
139.5, 139.4, 138.8, 132.9, 132.3, 130.0, 129.9, 128.9, 128.9,
123.3, 123.0, 115.0, 110.9, 110.8, 107.3, 107.1, 98.7, 97.0, 82.2,
82.2, 77.8, 77.1, 76.6, 75.2, 73.7, 73.6, 72.9, 72.2, 70.0, 69.0,
67.8, 57.5, 57.0, 56.8, 56.6, 56.3, 56.0, 55.0, 54.7, 52.7, 48.7,

Immunophilin Binding and Immunosuppressive Potency
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1775
48.5, 43.9, 43.4, 43.1, 40.3, 39.8, 39.2, 35.6, 35.6, 35.5, 34.6,
34.5, 33.6, 32.9, 32.7, 32.6, 30.4, 29.0, 27.5, 26.3, 26.2, 26.0,
24.6, 24.5, 24.2, 21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8, 15.6,
14.3, 14.2, 11.7, 9.8, 9.4; mass spectrum (FAB), 981 [M + K]+.
Anal. Calcd for C51H75N2O13F: C, 64.95; H, 8.02; N, 2.97.
Found: C, 64.82; H, 8.05; N, 2.91.
3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5 Hz), 2.74
(dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H), 2.33 (s,
3H), 1.67 (s, 1H), 1.65 (s, 2H), 1.63 (s, 1H), 1.61 (s, 2H), 1.01
(d, 2H, J ) 6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J )
7.5 Hz), 0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz),
0.88 (dd, 3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C
NMR (125 MHz) δ (rotamers) 213.3, 213.2, 196.1, 192.6, 168.9,
168.8, 168.6, 165.7, 164.6, 139.5, 138.6, 135.3, 133.6, 132.7,
132.2, 129.3, 129.1, 129.0, 123.2, 123.0, 119.7, 98.6, 97.0, 82.1,
82.0, 77.7, 77.3, 76.5, 75.1, 73.6, 73.6, 72.8, 72.2, 69.9, 68.9,
67.9, 57.4, 56.9, 56.7, 56.5, 56.2, 55.9, 54.9, 54.6, 52.6, 48.6,
48.3, 43.8, 43.4, 43.2, 40.2, 39.7, 39.1, 35.6, 35.5, 35.4, 34.5,
34.4, 33.6, 32.9, 32.6, 32.5, 30.3, 29.0, 27.5, 26.2, 26.1, 26.0,
24.5, 24.4, 24.1, 21.1, 20.8, 20.3, 19.4, 16.1, 15.9, 15.7, 15.6,
14.2, 14.0, 11.6, 9.7, 9.4; mass spectrum (FAB), 977 [M + K]+.
Anal. Calcd for C52H78N2O13‚H2O: C, 65.24; H, 8.42; N, 2.92.
Found: C, 65.13; H, 8.11; N, 2.93.
4-F lu or oa n ilin e-(32-Ascom ycin yloxya cet ic
a cid )
a m id e (28). 4-Fluoroaniline was coupled to AOAA (4) on a
0.88 mmol scale according to the method used for the synthesis
of 21. The silica pad (40 g, 70-230 mesh) was eluted with
CH2Cl2 (250 mL) followed by 2:1 hexane:acetone (300 mL).
Fractions containing the desired product were combined and
concentrated in vacuo to give 0.57 g of yellow foam which was
further purified by HPLC eluting with 2:1 hexane:acetone to
give 0.41 g of a semipure material. Further purification by
HPLC eluting with 5:2 CH2Cl2:CH3CN provided 28 (0.27 g,
32% yield): IR (KBr) ν 3425, 3310 (sh), 2920, 2850, 2820, 1740,
1695, 1643, 1610 (sh), cm-1 1H NMR (500 MHz) CDCl3
; δ
(rotamers 2:1) 9.63 (s, 0.33H), 9.62 (s, 0.67H), 7.58 (m, 2H),
7.03 (m, 2H), 5.34 (d, 0.67H, J ) 3.0 Hz), 5.22 (d, 0.33H, J )
3.0 Hz), 5.09-4.97 (mult, 2H), 4.87 (d, 1H, J ) 2.5 Hz), 4.62
(d, 1H, J ) 6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.25 (br
s, 1H), 4.24 (d, 0.67H, J ) 16.0 Hz), 4.23 (d, 0.33H, J ) 16.0
Hz), 4.00 (d, 0.67H, J ) 16.0 Hz), 3.99 (d, 0.33H, J ) 16.0
Hz), 3.98-3.87 (mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz),
3.68 (d, 0.67H, J ) 10.0 Hz), 3.61-3.08 (comp, 4.33H), 3.59
(mult, 1H), 3.55 (s, 3H), 3.39 (s, 2H), 3.38 (s, 1H), 3.35 (s, 1H),
3.31 (s, 2H), 3.03 (comp, 0.67H), 2.79 (dd, 0.67H, J ) 17.5, 4.5
Hz), 2.74 (dd, 0.33H, J ) 17.5, 4.5 Hz), 2.37-0.74 (comp, 26H),
1.69 (s, 1H), 1.67 (s, 2H), 1.64 (s, 1H), 1.61 (s, 2H), 1.01 (d,
2H, J ) 6.0 Hz), 0.98 (d, 1H, J ) 6.0 Hz), 0.95 (d, 2H, J ) 7.5
Hz), 0.92 (d, 1H, J ) 7.5 Hz), 0.89 (d, 2H, J ) 7.5 Hz), 0.88
(dd, 3H, J ) 7.5, 7.5 Hz), 0.85 (d, 1H, J ) 7.5 Hz); 13C NMR
(125 MHz) δ (rotamers) 213.5, 213.4, 196.1, 192.5, 169.0, 169.0,
168.7, 165.8, 164.7, 159.3 (d, 1J (CF) ) 237.5 Hz) 139.7, 138.8,
134.0, 132.9, 132.3, 129.0, 128.9, 123.3, 123.0, 121.4 (d, 3J (CF)
Ack n ow led gm en t. The authors wish to gratefully
acknowledge the contributions of the Abbott Analytical
Department for spectral data; Louis Seif and Bryan
Macri for conducting the hydrogenations; Loan Miller,
Morey Smith, and Michael Sheets for conducting the
in vitro experiments; and J anet Andrews, Patricia
Bretheim, Tom Fey, and Ruth Krause for conducting
the in vivo experiments.
Refer en ces
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FK506 has Peptidyl-prolyl Isomerase Activity but is Distinct
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2
) 12.5 Hz), 115.5 (d, J (CF) ) 25.0 Hz), 98.7, 97.1, 82.2, 77.8,
77.1, 76.6, 75.2, 73.7, 73.6, 72.9, 72.2, 70.0, 68.9, 67.9, 57.5,
57.0, 56.9, 56.6, 56.3, 56.0, 55.0, 54.7, 52.7, 48.7, 48.5, 43.9,
43.5, 43.1, 40.4, 39.8, 39.2, 35.7, 35.6, 35.5, 34.6, 34.5, 33.7,
32.9, 32.7, 32.6, 30.4, 29.0, 27.5, 26.3, 26.2, 26.0, 24.6, 24.5,
24.2, 21.2, 20.8, 20.5, 19.5, 16.2, 16.0, 15.8, 15.6, 14.3, 14.2,
11.7, 9.8, 9.5; mass spectrum (FAB), 981 [M + K]+. Anal.
Calcd for C51H75N2O13F: C, 64.95; H, 8.02; N, 2.97. Found:
C, 65.25; H, 8.21; N, 2.78.
4-Met h yla n ilin e-(32-Ascom ycin yloxya cet ic
a cid )
a m id e (29). A solution of 4-methylaniline (1.0 g, 9.6 mmol)
in dry THF (1 mL) was added dropwise to ethylmagnesium
bromide (1 M in THF, 9.6 mL) at 0 °C. The mixture was
stirred for 15 min and cooled to -78 °C, whereupon a solution
of 3b (0.8 g, 0.8 mmol) in dry THF (2 mL) was added dropwise.
After 1 h the mixture was added dropwise to a stirring biphasic
solution of 1 N H3PO4 (75 mL) and EtOAc (75 mL). The
organic layer was washed with saturated NaHCO3 solution
(75 mL) and brine (75 mL), dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was dissolved in CH2Cl2
and was then passed through a pad of silica gel (20 g) eluting
with CH2Cl2 (100 mL) and then 1:1 hexane:acetone (200 mL).
The filtrate containing the desired product was concentrated
in vacuo to give 0.87 g of dark yellow foam. Further purifica-
tion by HPLC eluting with 2:1 hexane:acetone provided 537
mg of semipure material. Final purification by HPLC eluting
with 4:1 CH2Cl2:CH3CN afforded pure 29 (0.39 g, 51% yield):
IR (KBr) ν 3440, 3300, 2960 (sh), 2925, 2870, 2820, 1740, 1695,
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1
1650 cm-1; H NMR (500 MHz) CDCl3 δ (rotamers 2:1) 9.52
(s, 0.33H), 9.50 (s, 0.67H), 7.49 (dd, 2H, J ) 9.5, 3.0 Hz), 7.14
(d, 2H, J ) 9.0 Hz), 5.34 (d, 0.67H, J ) 3.0 Hz), 5.22 (d, 0.33H,
J ) 3.0 Hz), 5.09-4.97 (mult, 2H), 4.85 (s, 1H), 4.62 (d, 1H, J
) 6.0 Hz), 4.45 (br d, 0.67H, J ) 15.0 Hz), 4.26 (s, 1H), 4.23
(d, 0.67H, J ) 16.0 Hz), 4.22 (d, 0.33H, J ) 16.0 Hz), 4.01 (d,
0.67H, J ) 16.0 Hz), 4.00 (d, 0.33H, J ) 17.5 Hz), 3.98-3.87
(mult, 1.33H), 3.75 (br d, 0.33H, J ) 14.5 Hz), 3.68 (d, 0.67H,
J ) 10.0 Hz), 3.58 (mult, 1H), 3.50-3.12 (comp, 4.33H), 3.55
(s, 3H), 3.39 (s, 2H), 3.38 (s, 1H), 3.35 (s, 1H), 3.30 (s, 2H),

1776 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Wagner et al.
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(21) Ascomycin (1b) was recrystallized from diethyl ether or aceto-
nitrile and is then stored as a crystalline solid. This material is
exceptionally difficult to redissolve in acetonitrile unless it is
returned to a solid “glass-like” state by dissolution in CH2Cl2
followed by removal of the solvent in vacuo. Preventing rapid
crystallization upon addition of the acetonitrile is made possible
only by premixing 1b with benzyl iodoacetate.
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(22) Unless the Ag2O is added in small portions at 0 °C over the
indicated time period, a vigorous exothermic reaction ensues
which results in immediate and complete decomposition of the
ascomycin (1b). It is presumed that iodide is released under
these uncontrolled conditions, and this then adds to the C-9
carbonyl, promoting the well-documented benzylic acid re-
arrangement.
J M960066Y